Water-solubilized, cap-stabilized, helical polyalanines: Calibration standards for NMR and CD analyses

Published on Web 01/22/2005

Water-Solubilized, Cap-Stabilized, Helical Polyalanines:

Calibration Standards for NMR and CD Analyses

Bjo¨rn Heitmann, Gabriel E. Job, Robert J. Kennedy, Sharon M. Walker, and

Daniel S. Kemp*

Contribution from the Department of Chemistry, Room 18-296, Massachusetts Institute of

Technology, Cambridge, Massachusetts 02139

Received July 15, 2004; E-mail: kemp@mit.edu

Abstract: NMR and CD studies are reported for two length series of solubilized, spaced, highly helical

â

polyalanines that are N-capped by the optimal helix stabilizer Asp-Hel and C-capped by â-aminoalanine

beta and that are studied in water at 2 °C, pH 1-8. NMR analysis yields a structural characterization of the

â

peptide Ac^âAspHelAla₈betaNH₂and selected members of one AspHelAla_nbeta series. At pH > 4.5 the

^âAspHel cap provides a preorganized triad of carboxylate anion and two amide residues that is

complementary to the helical polyalanine N-terminus. The C-terminal â-aminoalanine assumes a helix-

stabilizing conformation consistent with literature precedents. H(N)CO NMR experiments applied to capped,

uniformly ¹³C- and ¹⁵N-labeled Ala₈and Ala₁₂peptides define Ala_nhydrogen bonding signatures as R-helical

without detectable 3₁₀character. Relative NHfND exchange rates yield site protection factors PF_ithat

define uniquely high fractional helicities FH for the peptide Ala_nregions. These Ala_ncalibration series,

studied in water and lacking helix-stabilizing tertiary structure, yield the first ¹³C NMR chemical shifts, ³J_HNHR

coupling constants, and CD ellipticities [θ_Molar]_λ,ncharacteristic of a fully helical alanine within an Ala_ncontext.

CD data are used to assign parameters X and [θ]_λ,∞, required for rigorous calculation of FH values from

CD ellipticities.

Introduction

45, in water, but these lack conformational homogeneity.

Fractional helicities⁶FH for the Ala_nregion are substantially

Alanine is the simplest strongly helix-forming R-amino acid,

and polyalanines are natural choices for modeling helical

structure and energetics.¹Bio-relevant tests of modeling predic-

tions require helicity data for fully helical Ala_npeptides in

aqueous solution. A definitive X-ray fiber diffraction structure

of helical polyalanine in the solid state was reported nearly forty

years ago,²but up to now, comparable structural studies in water

have been unavailable. Low Ala_nsolubilities and high aggrega-

tion have long been addressed by triblock constructs that flank

central polyalanines by solubilizing N- and C-terminal peptide

caps.³

As well as inhibiting formation of Ala_nâ-sheet aggregates,⁴

an ideal pair of caps must suppress extension of the helix beyond

the boundaries of the Ala_nregion as well as interactions of

charged solubilizers with the helix. Heteropeptide constructs

shown in Figure 1a⁵solve these problems and allow analysis

of intrinsic properties of simple helical Ala_npeptides, 6 e n e

less than 1.0,⁵since the completely helical conformation

equilibrates with nonhelical and partially helical conformations

of comparable stability.⁷In the first part of this report we

characterize the first water-soluble, maximally helical Ala_n

length series and use it to assign structure and fundamental NMR

parameters for hydrated, R-helical alanines.

How is maximal helicity achieved? Our research strategy was

defined by Lifson-Roig modeling.⁸If a homopeptide derived

from a residue with a high helical propensity is N- and C-capped

by strong helix stabilizers, the completely helical conformation

is selectively stabilized and can dominate the manifold. The

peptide Ac-^âAspHel-Ala₈-beta-NH₂, with the N-cap ^âAsp-Hel⁹

and the C-cap â-aminoalanine ≡ beta,¹⁰was previously shown

from NH f ND exchange-derived site protection factors PF_is¹¹

(5) (a) Miller, J. S.; Kennedy, R. J.; Kemp, D. S. Biochemistry 2001, 40, 305-

309. (b) Miller, J. S.; Kennedy, R. J.; Kemp, D. S. J. Am. Chem. Soc.

2002, 124, 945-962. (c) Kennedy, R. J.; Tsang, K.-Y.; Kemp, D. S. J.

Am. Chem. Soc. 2002, 124, 934-944.

(6) For each peptide under particular experimental conditions, FH is defined

as the ratio of helical R-carbons to all Ala_nR-carbons; its range is 0 to 1.

Owing to charge repulsion, Lys_mregions are nonhelical.

(7) (a) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526-535. (b)

Zimm, B. H.; Doty, P. Iso, K. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 1601-

1607. (c) Lifson, S.; Roig, A. J. Chem. Phys. 1961, 34, 1963-1974.

(8) Kemp, D. S. HelV. Chim. Acta 2002, 85, 4392-4423.

(9) Maison, W.; Arce, E.; Renold, P.; Kennedy, R. J.; Kemp, D. S. J. Am.

Chem. Soc. 2001, 123, 10245-10254.

(10) Deechongkit, S.; Tsang, K.-L.; Renold, P.; Kennedy, R. J.; Kemp, D. S.

Tetrahedron Lett. 2000, 41, 9679-9683.

(11) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins: Struct., Funct.,

Genet. 1993, 17, 75-86.

(1) (a) Wieczorek, R.; Dannenberg, J. J. J. Am. Chem. Soc. 2003, 125, 8124-

8129. (b) Mahadevan, J.; Lee, K.-H.; Kuczera, K. J. J. Phys. Chem. B 2001,

105, 1863-1876. (c) Levy, Y.; Jortner, J.; Becker, O. M. Proc. Natl. Acad.

Sci. U.S.A. 2001, 98, 2188-2193. (d) Lee, S.-H.; Krimm, S. Biopolymers

1998, 46, 283-317.

(2) Arnott, S.; Dover, S. D. J. Mol. Biol. 1967, 30, 209-212.

(3) (a) Gratzer, W. B.; Doty, P. J. Am. Chem. Soc. 1963, 85, 1193-1197. (b)

Ingwall, R. T.; Scheraga, H. A.; Lotan, N.; Berger, A.; Katchalski, E.

Biopolymers 1968, 6, 331-368.

(4) (a) Forood, B.; Pe´rez-Paya´, E.; Houghten, R. A. Blondelle, S. E. Biochem.

Biophys. Res. Commun. 1995, 211, 7-13. (b) Blondelle, S. E.; Forood,

B.; Houghten, R. A.; Pe´rez-Paya´, E. Biochemistry 1997, 36, 8393-8400.

9

1690

J. AM. CHEM. SOC. 2005, 127, 1690-1704

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

Figure 1. (a, b) Schematics depicting peptide sequences for spaced, solubilized polyalanines. (a) Color code: red, solubilizing polylysine regions; yellow,

spacers that confine helical structure to the green Ala_nregion and isolate it from interactions with the charged Lys_mregions. (b) Added are the helix-

â

stabilizing N-capping regions AspHel, blue, and the helix-stabilizing and terminating C-cap, beta ≡ â-aminoalanine, purple. Spacing elements Sp include

combinations of ^t^L-tert-leucine, Inp ≡ 4-carboxypiperidine; Acc ≡ trans-4-aminocyclohexanecarboxylic acid. (c) Depiction of the helical hydrogen bonding

connectivity of the ^âAspHel-Ala_n-beta sequence, as demonstrated in this report.

to have a FH z 0.95.^5cIn this report we generalize this result

to members of ^âAspHel-Ala_n-beta length series, characterizing

their Ala_nregion FH values by NH f ND exchange. These

provide a hitherto missing calibration for CD-based FH

assignments.^5b,12

This study is timely. It introduces model systems that may

be used to confirm predictions of recent polyalanine modeling

experiments;¹it takes a key step toward improving the accuracy

and precision of CD-based helicity assignments;¹³and it may

provide insight into sequences and experimental conditions that

predispose or inhibit sheet or helix formation by polyalanine

peptide sequences of current relevance to materials science¹⁴

and to medicine.¹⁵

familiar eq 1. In subsequent sections, we use NMR and CD

data to assign this minimum length.

[θ]_λ,n) [θ]_λ,∞(1 - X/n) ) [θ]_λ,∞- [θ]_λ,∞(X/n) (1)

[θ]_λ,nis the molar per residue ellipticity at λ nm of a peptide of

length n with FH 1.0; X, with dimensions of length in residues,

corrects for capping effects; [θ]_λ,∞is the convergent core

ellipticity

Design of N- and C-Capped Peptides Containing a

â

Central Sequence AspHel-Ala_n-beta for NMR and CD

Spectroscopic Roles: Peptide 1 and the Peptide Series

2 and 3

â

An Overview of NMR and CD Structural Issues

NMR and CD experiments require different AspHel-Ala_n-

beta peptide series. The CD series must span a large range of

n, and the lengths of the Lys_mcaps must be sufficient to inhibit

aggregation. NMR structural studies require spectra that are

A key question underlies both NMR and CD studies. Do the

N- and C-caps of the ^âAspHel-Ala_n-beta series selectively

enhance the mole fractions of the completely helical Ala_n

conformations without globally distorting their structures? In

other words, can one treat the capping effect as a structural

perturbation, confined to terminal regions? The intrinsic structure

of an R-helix renders cap-initiated global distortions implausible.

The helical backbone defines a cylinder of fixed diameter, and

all its intramolecular hydrogen bonds, van der Waals contacts,

and nonpolar interactions occur locally, between pairs of

residues belonging to contiguous three-residue loops. For n

larger than an experimentally assigned minimum length, and

for an ideal fully hydrated Ala_nhelix, the properties of core

residues that are isolated from the terminal regions should

converge to limiting cap-independent values. This principle

underlies CD-based quantitation of FH¹⁶that relies on the

1

dominated by Ala resonances, with minimal H, ¹³C, and ¹⁵N

resonance overlaps. The initial NMR structural assignments

employed the previously studied Ala₈peptide 1, which lacks

solubilizing regions.^5c

Peptides of series 3 were designed to meet the needs of CD

calibrations.⁵The Trp residue, which is isolated from the helical

region, was required for accurate concentration determination,

and the ten Lys residues were chosen from previous studies.^5b

Although PF_imeasurements and other NMR characterization

experiments were carried out on representative members of this

(12) (a) Wallimann, P.; Kennedy, R. J.; Kemp, D. S. Angew. Chem., Int. Ed.

1999, 38, 1290-1292. (b) Wallimann, P.; Kennedy, R. J.; Miller, J. S.;

Shalongo, W.; Kemp, D. S. J. Am. Chem. Soc. 2003, 125, 1203-1220.

(13) (a) Chakrabartty, A.; Kortemme, T.; Baldwin, R. L. Protein Sci. 1994, 3,

843-852. (b) Park, S. H.; Shalongo, W.; Stellwagen, E. Biochemistry 1993,

32, 7038-7053. (c) Myers, J. K.; Pace, C. N.; Scholtz, J. M. Biochemistry

1997, 36, 10923-10929.

(14) (a) Nakazawa, Y.; Asakura, T. Y. Macromolecules 2002, 35, 2393-2400.

(b) Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.

(15) (a) Baxter, S. W.; Choong, D. Y. H.; Ecles, D. M.; Campbell, I. G. Cancer

Epidemiol., Biomarkers PreV. 2002, 11, 211-214. (b) Bruneau, S.; Johnson,

K. R.; Yamamoto, M.; Kuroiwa, A. Duboule, D. DeV. Biol. 2001, 237,

345-353. (c) Perutz, M.; Pope, B. J.; Owen, D.; Wanker, E. E.; Scherzinger,

E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5596-5600.

(16) (a) Chen, Y.-H.; Yang, J. T.; Martinez, H. M. Biochemistry 1972, 11, 4120-

4131. (b) Woody, R. W.; Tinoco, I., Jr. J. Chem. Phys. 1967, 46, 4927-

4945. (c) Johnson, W. C., Jr.; Tinoco, I., Jr. J. Am. Chem. Soc. 1972, 94,

4389-4390.

3

series, assignments of J_HNHRcoupling constants and residue-

specific PF_ivalues were complicated by anomalous splitting

and broadening of Ala NH resonances, attributable to the Inp

spacers. Inp itself is achiral, but its simple N-acyl derivatives

contain a chiral center, Figure 2, and the corresponding series

3 peptides, which contain a pair of Inp₂functions, are diaster-

eomeric mixtures. The expected NH peak widths and multiplici-

ties are restored if Inp₂is replaced in series 2 by a single achiral

Acc spacing element derived from trans-4-aminocyclohexane-

carboxylic acid. This series, patterned on structure 1, was

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1691

A R T I C L E S

Heitmann et al.

Figure 2. (a) Depiction of a pair of isoenergetic mirror image N-acyl-Inp

functions; owing to amide resonance, these are in slow equilibrium on the

NMR time scale at ambient temperatures. A pair of diastereomers is

generated by each Inp function, accounting for the multiplicities and line

broadening observed for Ala_n¹H NH resonances of series 3 peptides. (b)

Depiction of the achiral, rigid Acc used as a spacing element for series 2

peptides. Derived from trans-4-aminocyclohexane carboxylic acid, this

element joins peptide regions by secondary amide bonds that assume the

s-trans conformation. Modeling estimates the Acc distance between its 1-Cd

O and 4-NH atoms as 6.7 Å.

Figure 3. Three conformational states of a peptide conjugate of the N-acyl-

Hel N-cap. On the NMR time scale in water at 2 °C, the (ts) and (te) states

equilibrate and appear as a single set of resonances; owing to slow (c)-(t)

amide rotation, the (cs) and composite (ts) + (te) states appear as distinct

resonances. The (te) state stabilizes the helix, but the (cs) and (ts) states do

not. For the highly helical peptides of this report, the (te) conformation is

dominant.^5c

designed for both CD and NMR studies. To avoid aggregation

we fixed the upper length limit for series 2 peptides at 16. For

pairs of members of series 2 and 3 with identical Ala_nregions,

cap-corrected CD spectra are equivalent within measurement

error, as are FH values calculated from PF_imeasurements.

Application of AUC under CD and NMR measurement

conditions to selected peptides that span the full length range

of series 2 and 3 demonstrated absence of detectable aggrega-

tion.¹⁷A preliminary test of NH f ND exchange rates in D₂O

revealed that all non-Ala NH resonances exchange at rates

expected for solvent-exposed protons. This result implies that

hydrogen-bonded or solvent-shielded NH functions appear solely

within the Ala_nregions for peptides of each series.

also form one or more strong hydrogen bonds to the three helical

C-terminal carbonyl oxygens.¹⁰

Capping Region NMR Assignments for Peptides 1 and

2; Conformations and Interactions with the Ala₈Region

â

for AspHel and beta; Helical Stabilization by an

â

Ionized Asp Function

Initial 600 MHz NMR structural studies were carried out on

peptide 1, Ac-^âAspHel-Ala₈-beta-NH₂, in aqueous solution at

A brief review of helical structure introduces the next section

which presents NMR evidence for cap conformations and

1

2 °C, pH 3. Analysis of a H NMR spectrum together with

â

1

interactions characteristic of AspHel-Ala_n-beta peptides. An

TOCSY and ROESY spectra allowed assignment of all H

R-helix, Figure 1c, is spanned by three chains of successively

hydrogen-bonded amides. A central helical amide at sites i and

(i + 1) forms a donor H-bond to the amide CO of site (i - 3)

and an acceptor H-bond to the amide NH of site (i + 4). Each

of the three amides at the two helix ends forms only one

intrahelical H-bond and carries an unsatisfied H-bonding

valence. At each terminus, local amide dipoles sum to an

experimentally demonstrable partial charge, positive at the

N-terminus, negative at the C-terminus.¹⁸Native structures of

globular proteins often contain side chain functions that

terminate helices by providing stabilizing terminal charges as

well as missing hydrogen bond donors or acceptors.¹⁹Helix

stabilizing N- and C-caps are designed to incorporate these

features.

resonances; no evidence for tertiary structure was detected. The

major conformer was assigned a (t)-state structure, analogous

to that previously characterized for Ac-Hel-peptide conjugates,

Figure 3.²⁰The spectra show eight resolved alanine NH

resonances, linked by a continuous ladder of N,N(i, i + 1) cross-

peaks. The Ala-1 resonance was assigned by cross-peaks that

tie it to single Ala and to the Hel function. The NH resonance

assigned to Ala-8 showed a pair of (i, i + 1) cross-peaks with

the NH resonance of Ala-7 and with the backbone NH of beta;

the latter in turn showed an (i, i + 1) cross-peak with one of

the two resonances of the C-terminal NH₂. These are consistent

with the expected Ala₈helical structure, terminated by the N-

and C-caps.

A left-handed helical conformation is assigned for beta,

Figure 4a, from a pair of cross-peaks between the resonance

corresponding to the R-proton of Ala-6 and both the R and one

of the â-protons of beta, together with a cross-peak between

one of the C-terminal NH₂protons and the â-proton of Ala-8.

Similar conformations are reported for R-helix C-termination

by Nagarajaram et al. from analysis of Asn and Gly examples

from the protein X-ray crystallographic database.²¹

â

The AspHel N-cap provides an anion that can stabilize the

local N-terminal helical dipole and act as an H-bond acceptor.

The anion and two more H-bonding amides are preorganized

to match orientations of the N-terminal helical NHs, Figure 1c.⁹

The C-cap beta provides an oriented positive charge, which may

(17) Although the series 2 peptides (n e 16) are unaggregated, longer members

of this series have not been studied, and the aggregation-free upper limit

of n for this series at millimolar concentrations in water is currently

unknown.

(18) Tidor, B. Proteins: Struct., Funct., Genet. 1994, 19, 310-323.

(19) Aurora, R.; Rose, G. D. Protein Sci. 1998, 7, 21-38.

(20) Kemp, D. S.; Allen, T. J.; Oslick, S. L. J. Am. Chem. Soc. 1995, 117,

6641-6657.

(21) Nagarajaram, H, A.; Sowdhamini, R.; Ramakrishnan, C.; Balaram, P. FEBS

Lett. 1993, 321, 79-83.

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1692 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

Figure 4. Computer-generated stereodiagrams consistent with ROE data. (a) The Ala₈-beta C-terminus. (b) The ^âAspHel-Ala₈N-terminus. Color code:

black, Ala₈bonds and atoms; blue, beta and ^âAspHel atoms and bonds; red, Ala-Ala H-bonds; orange, Ala₈-beta H-bonds; green, Ala₈-^âAspHel H-bonds.

Structures were generated by Quanta (Molecular Simulations, Inc.).

TOCSY assignments of CH chemical shifts and connectivities

for resonances attributable to the Hel function correspond closely

to a previously reported NMR characterization of the Hel

function of AcHel-Ala₆-OH.²⁰The R-CO-Hel function is

remarkably rigid, but it embodies three degrees of two-state

conformational freedom. Its tertiary N-acylamido group can

assume either (c) or (t) orientations; the ψ dihedral angle of its

peptide-linked carboxamido function can assume either helical

or γ-turn orientations, and its eight-membered lactam can

assume either helix-initiating (e) or noninitiating (s) conforma-

tions. All are defined for the major NMR conformation. A cross-

peak links the resonances assigned to protons at C₁₂and the

â-CH₂of the Asp function and establishes the orientation of

the Asp-Hel amide bond as (t). A cross-peak linking Hel C-9

and C-13 proton resonances establishes the orientation of the

eight-membered ring of the Hel function as (e). Three cross-

peaks are detected between the resonance of the NH proton of

Ala-1 and resonances corresponding to C-6, C-7, and C-8 Hel

protons. These establish the orientation of the Hel C-5 ψ dihedral

angle as helical.²²

with those assigned for peptide 1. These assignments correspond

to a helix-nucleating (te) state of AspHel, Figure 4b. Reso-

â

nances belonging to Ac^âAsp exhibit intraresidue cross-peaks,

but none defined contacts with the Ala_nregion. The pH

dependences of NH resonances of peptide 1 and selected

members of series 2 are more informative. If the pH is varied

from 1.5 to 8.5, three correlated changes in chemical shift are

observed: 0.45 ppm for the resonance of the NH function of

Ac-^âAsp, 0.19 ppm for the NH resonance of Ala-1, and 0.13

ppm for the NH resonance of Ala-2. Ionization of the carboxylic

acid of Ac-^âAsp is expected to have a large chemical shift effect

on its own NH, since these groups are proximate. The observed

shift changes for Ala₁and Ala₂NH resonances imply that in

the ionized structure the carboxylate anion is also proximate to

these protons.

The titration curve for each chemical shift yields pK_avalues

of 3.29 in water, 2 to 50 °C, that correspond to ionization of

the Asp R-carboxylic acid (Figure 5). This pK_avalue, calculated

from local chemical shifts of single NH protons, is also

assignable from the pH dependence of [θ]₂₂₂, which reflects

the global anion-dipole helix stabilization. Ionization results

in a ca. 20% increase in CD fractional helicity for Ala_npeptides,

n ) 7 to 9, confirming our postulated role for the carboxylate

TOCSY, NOESY, and 1D ¹H NMR spectra were also

measured at pH 4.5, 2 °C in aqueous buffer for members of the

peptide series 2 Ac-^âAspHel-beta-AccLys₂TrpNH₂, n even,

8-16. Resonances of the capping regions of these peptides and

their NOE cross-peaks with end regions of Ala_nwere consistent

â

anion of N-cap AspHel.⁹

Resonances attributable to a minor conformation can also be

detected at low pH in NMR spectra of Ala_npeptides. These are

assigned, Figure 3, to the previously documented weakly helical

(22) Cammers-Goodwin, A.; Allen, T. J.; Oslick, S. L.; McClure, K. F.; Lee, J.

H.; Kemp, D. S. J. Am. Chem. Soc. 1996, 118, 3082-3090.

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A R T I C L E S

Heitmann et al.

to the literature database, protein or peptide chemical shift data

can be used to predict local regions of helical structure.²⁴Helical

1

backbone HN chemical shifts are sensitive to hydrogen bond

length and strength, solvent exposure, deviations from helical

rod structures, and residue positioning within either the N- or

the C-terminus. Experimental constancy of ¹HN shifts thus

provides a sensitive test for Ala_nstructural convergence.

Resonances of eight Ala ¹HN residues belonging to the two

end regions of members of series 2 exhibit distinctive chemical

shifts and are consistently resolved. At the N-terminus for the

series members with n ) 10 to 16, chemical shifts for Ala-1

1

appear in the unusual range 7.48 ( 0.02 ppm, but the HN

chemical shifts for Ala-2 and 3 appear within a more normal

helical range of 8.37 ( 0.02 ppm. Chemical shifts for Ala-4

fall in the distinctive range 8.11 ( 0.02 ppm, which may reflect

a local structural transition from hydrogen bonding acceptor

oxygens of tertiary amides of the Hel function to the oxygen of

the secondary amide at the Hel-Ala_njunction.

Figure 5. Titration of the ^âAsp carboxylic acid of Peptide 1 in water at 2

°C, monitored by changes in the chemical shift of the first alanine NH

function (similar curves result from monitoring chemical shifts of the second

alanine NH or the Asp NH). The curve is calculated from respective limiting

chemical shifts for the conjugate acid (7.314 ( 0.003) and base (7.497 (

0.002) and from an assigned pK_avalue at 2 °C of 3.27 ( 0.035; at 25 and

49 °C, respective pK_avalues of 3.31 ( 0.02 and 3.27 ( 0.02 were assigned.

Resonances for ¹HNs at the (n - 3) and (n - 2) sites appear,

respectively, at 8.09 ( 0.01 ppm and 8.08 ( 0.01 ppm, and

resonances at the (n - 1) and n sites appear, respectively, at

7.93 ( 0.02 and 7.95 ( 0.02 ppm. From the PDB, downfield

(cs) state for which the N-cap is a poor helix stabilizer. We

have previously introduced t/c, the ratio of integrated areas of

(c) and (t) state resonances, as a quantitative measure of helicity

and have shown that any experimental variable that increases

helical stability also increases t/c.²⁰Consistent with this assign-

ment, the relative intensity of the resonances of the minor

conformations of the peptides 1 and 2 is substantially decreased

by helicity-increasing variable changes: lower temperatures, a

pH increase above 3.4, an increase in Ala_nlength, and addition

of trifluoroethanol. The CD and PF_imeasurements of this study

were conducted in aqueous solutions at 2 °C and above pH 4.5;

under these conditions, t/c is large.²³

From the evidence of this section, the C-cap beta has been

shown to interact locally with the C-terminal loop of the Ala_n

sequence, terminating the helical sequence¹⁰and optimally

positioning the positive charge of beta for helix stabilization.

Above pH 4.5, the ionized N-cap ^âAspHel assumes the

previously characterized (te) conformation that allows two amide

oxygens of the Hel function to form donor hydrogen bonds with

NHs of the second and third amides of the Ala_nregion. The

carboxylate anion of the ^âAsp function is positioned to hydrogen

bond with the first amide NH of the Ala_nregion, allowing its

charge to stabilize the N-terminus of the Ala_nhelical conforma-

tion initiated by the ^âAspHel cap. The peptides are unaggregated

in water and show no evidence of tertiary structure; NH

hydrogen exchange is slow within the Ala_nregion but rapid for

all other residues; and NMR evidence defines the structures of

the ^âAspHel and beta caps and demonstrates their local

interactions with the Ala_nregion. As summarized in Figure 4a,b,

these observations demonstrate peptide-cap interactions of these

peptides that are consistent with an Ala_nR-helix and confined

to its first three and last three residues.

1

shifts for HNs at a helical N-terminus and upfield shifts at a

C-terminus have been documented and attributed to a helix

dipole effect.²⁵This effect as well as helical fraying at the

C-terminus of these peptides may account for these systematic

site-specific changes. Corresponding chemical shifts²⁶for the

Ala₈peptide of series 2 are qualitatively similar. The NMR

properties of the eight N-terminal and C-terminal residues of a

series 2 Ala_npeptide sequence, n ) 8 or larger, show

characteristic site-dependent values that are independent of the

length n.

The ¹HN chemical shifts for resonances found at all remaining

sites 5 to (n - 4) within the five peptides of series 2, n ) 10-

16 are unresolved, with a mean value of 8.21 ( 0.02 ppm. For

1

the central n - 8 residues of these peptides, these H NH

chemical shifts demonstrate convergence, consistent with their

structural isolation from capping regions.

The chemical shifts of ¹³CdO and ¹³CR functions correlate

with FH values²⁷and are reliable reporters of helical structure

within protein sequences.²⁵Invariably the largest ¹³C chemical

shifts are seen for helices. The reported averages for protein-

derived values of ¹³CdO chemical shifts of alanine residues

within respective â-strand, coil, and helical regions are 175.0,

177.6, and 179.6 ppm; the values of alanine ¹³CR chemical shifts

are 50.3, 52.4, and 54.7 ppm. Coil values are in good agreement

those observed for short peptides (¹³CdO, 177.6 ppm; ¹³CR,

52.2 ppm), and the helical values are consistent with values

assigned from a recent study of a highly helical alanine-rich

peptide at 0 °C in water (¹³CdO, 180 ppm; ¹³CR, 54.0 ppm).

To assign ¹³C chemical shifts to members of Ala_nseries and

to facilitate measurement of other key helicity parameters, three

peptides were synthesized containing alanine residues labeled

Assignment of H and ¹³C Chemical Shifts to Alanine

Residues of the Ala_nRegion

1

(24) Wishart, D. S.; Sikes, B. D., Methods Enzymol. 1994, 239, 363-392.

(25) Wishart, D. S.; Richards, F, M.; Sikes, B. D. J. Mol. Biol. 1991, 222, 311-

333.

(26) The following ¹HN chemical shifts (ppm) are seen for the series 2 Ala₈

peptide: site 1, 7.50; site 2, 8.29; site 3, 8.26; site 4, 8.00; site 5 (n - 3),

8.02; site 6 (n - 2), 8.00; sites 7 and 8 (n - 1, n), 7.90.

(27) (a) Shalongo, W.; Dugad, L.; Stellwagen, E. J. Am. Chem. Soc. 1994, 116,

2500-2507. (b) Werner, J. H.; Dyer, R. B.; Fesinmeyer, R. M.; Andersen,

N. H. J. Phys. Chem. B 2002, 106, 487-494.

Assignment of protein secondary structures from NMR

chemical shifts has received much recent attention. By reference

(23) As detailed in the Experimental Section, for pH values greater than 4.5,

and for Ala_nlengths of at least six residues, t/c values g 50 are expected.

9

1694 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

above the peptide values, with pairs linked with N-terminal Ala

residues appearing at the upper right of the Ala₁₂sequence. The

three pairs that lie at or in the pink zone correspond to residues

within the Ala₁₂C-terminal loop. Like the site dependence of

¹H NH chemical shift values, this pattern is consistent with

progressive helical fraying toward the Ala₁₂C-terminus. For

the core Ala-5 and Ala-6 residues of this sequence, the carbonyl

carbon shifts are identical, 180.90 ppm, as are R-carbon chemical

shifts, 54.87 ppm; these lie at the upper end of values assigned

from protein structures. These are the convergent values for ¹³

C

chemical shifts of alanine residues within core helical regions

of polyalanines in water at 2 °C.

Chemical shift data can be diagnostic for helical structure,

but detailed conformational assignments are usually based on

NOEs, which for Ala_npeptides require site-labeled peptides.²⁹

Even with a definitive labeled series, 3₁₀-R structural distinctions

can be problematic. As noted recently,³⁰a more reliable

alternative is available. The modified H(N)CO NMR experiment

of Cordier and Grzesiek^31,32yields ¹⁵Ns¹³CdO scalar couplings

3h

J

that directly define helical amide-amide ¹⁵NsH‚‚‚Od

NC′

¹³C hydrogen bonds.

The schematic of Figure 1a identifies pairs of alanine residues

of a labeled Ala₈peptide with ¹H and ¹³C nuclei that are

Figure 6. Correlation of ¹³CdO and ¹³CR chemical shifts for the labeled

series 2 Ala₈peptide (water, pH ca. 3) and the Ala₁₂peptide (water, pH >

4.5). The red dashed lines are derived from the protein database and are an

interface between helix and coil values.^24,25The pink area defines the

average, within one SD unit, of the ¹³CdO values reported for a highly

helical peptide.²⁷Pairs of Ala chemical shift values for the Ala₁₂peptide

appear as cyan squares (owing to overlap, only 10 pairs of values are

plotted). Pairs for the (te) state of the Ala₈peptide appear as yellow squares;

the small green square corresponds to the chemical shift pair of the Ala-1

(cs) state. The large green square corresponds to the average, within 1 SD,

of the remaining (cs) state resonances. (Because of weak signal intensities,

residue assignments could not be made.)

3h

expected to exhibit scalar R-helical

J

couplings. For a

NC′

peptide with n labeled alanines, the modified H(N)CO NMR

experiment should exhibit n - 4 cross-peaks that correspond

1

to hydrogen bonds that link ¹³CdO at each site i with a H-

¹⁵N at site (i + 4). For the series 1 Ala₈peptide in which all

Ala residues are uniformly labeled with ¹³C and ¹⁵N, the

3h

following four residue pairs should show a

J

: 1 and 5, 2

NC′

and 6, 3 and 7, 4 and 8.³³Figure 7a shows results of applying

the 2D H(N)CO experiment to this peptide. (It should be noted

that the seven purple i, i + 1 cross-peaks allow assignment of

covalent amide connectivities and the ¹³CdO chemical shifts

with ¹³C and ¹⁵N. Their ¹³CdO chemical shifts were assigned

from a 2D version, H(N)CO, of a standard HNCO experiment,²⁸

which correlates a ¹³CdO chemical shift for a residue at

sequence site i with the previously assigned ¹HN chemical shift

at sequence site i + 1. An unambiguous assignment requires

of Figure 6.) The four green R-helical i, i + 4 cross-peaks

3h

correspond to

J

scalar couplings that link residues 1 with

NC′

5 and 2 with 6; a confluent region defines the couplings that

link residues 3 with 7 and 4 with 8. These verify the R-helical

structure.

Figure 7b shows the corresponding 2D spectrum for the series

2 Ala₁₂peptide in which residues 7-12 were labeled. An

1

that no resonance overlaps occur, within either the H or the

¹³C set. This condition was met by the labeled Ala₈peptide 1,

but it is unlikely to be met for longer members of series 2. All

1

Ala resonances of the series 2 Ala₁₂peptide were assigned H

R-helical hydrogen bonding pattern requires that the spectrum

and ¹³C chemical shifts by studying two peptides, one doubly

labeled at alanines 1-6, the other at alanines 7-12.

3h

should reveal two R-helical

J

scalar couplings that link

NC′

residues 7 with 11 and 8 with 12. Both of these are evident.

Application of the same experiments to the Ala₁₂peptide with

labeled sites Ala-1 through Ala-6 confirmed the presence of

R-helical hydrogen bonds that link amides of residue 1 with 5

and 2 with 6 (spectrum not shown).

For the labeled Ala₁₂peptide of Figure 7b, the presence of

significant contributions of conformations with 3₁₀helical

structure should be signaled by appearance of cross-peaks

corresponding to hydrogen bonds that link residues 7 with 10,

Figure 6 shows the results of measurement of ¹³C chemical

shifts observed for the ¹³CdO and ¹³CR resonances at 8 °C in

aqueous buffer for the labeled peptides. The plot includes red

dotted lines that define the literature boundary between helix

and coil for alanine residues of the PDB and a pink region that

corresponds to the range of ¹³CdO chemical shifts reported for

the alanine-rich peptide.²⁷Our Ala₈peptide was studied at pH

â

3 at which the helix-stabilizing AspHel cap functions subop-

timally; as a result both its (te) and (cs) state resonances can be

detected. With the exception of the borderline resonance that

corresponds to the C-terminal alanine, the former lie well within

the expected helical regions, and the latter lie close to random

coil values.

(29) Kemp, D. S.; Allen, T. J.; Oslick, S. L.; Boyd, J. G. J. Am. Chem. Soc.

1996, 118, 4240-4248.

(30) Dehner, A.; Planker, E.; Gemmecker, G.; Broxterman, Q. B.; Bisson, W.;

Formaggio, F.; Crisma, M.; Toniolo, C.; Kessler, H. J. Am. Chem. Soc.

2001, 123, 6678-6686.

(31) Dingley, A.; Grzesiek, S. J. Am. Chem. Soc. 1998, 120, 8293-9297.

(32) Cordier, F.; Grzesiek, S. J. Am. Chem. Soc. 1999, 121, 1601-1602.

(33) For the N- and C-capped Ala_npeptides of this study, one could in principle

demonstrate four more hydrogen bonds between the caps and the Ala_n

region if the appropriate ¹³CdO labeled ^âAspHel and of ¹⁵N labeled beta

were available.

The Ala₁₂peptide was studied at pH > 4.5. The majority of

its (te) chemical shift pairs fall within the protein helical region,

(28) Grzesiek, S.; Bax, A. J. Magn. Reson. Ser. 1992, 96, 432-440.

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1695

A R T I C L E S

Heitmann et al.

8 with 11, and 9 with 12. In the figure, the sites of these potential

cross-peaks are indicated by gold crosses. Even for the 7, 10

link, which would appear without overlap, no cross-peak is

evident. This issue is explored more rigorously in Figure 7c,

which shows optimized 1D slices measured at the ¹³CdO

chemical shifts of the Figure 7b green cross-peaks. No 3₁₀cross-

peak can be detected within the signal-to-noise limits of the

experiment.

If an amino acid sequence has been independently established

as R-helical, the ³J_HNHRcoupling constant defines the φ dihedral

angle, which for an R-helix can fall in the range of -30 to -80°.

An R-helix has a parametric dihedral constraint, φ + ψ )

-103°, allowing ψ to be defined from the assigned φ.³⁴The φ

value for the Pauling-Corey helix³⁵is -47 °, and that assigned

by Arnott and Dover²from an X-ray fiber diffraction study of

polyalanine is -57.4°, but the average value for helices within

the PDB is -62 ( 7°.³⁶

3

The J_HNHRcoupling constants were assigned to the helical

alanine residues in the three peptides from an E.COSY HNCA

experiment.³⁷The average for the twelve amino acids of the

series 2 A₁₂peptide in water at 2 °C is 3.4 Hz (SD 0.5), and

the pair of core alanines at sites 5 and 6 showed identical values

of 3.0 Hz. Fits of the latter value to the Karplus equation using

parameters of Vuister,³⁸Pardi,³⁹and Ludvigsen⁴⁰yield respec-

tive φ values of - 50.4°, -48.6°, and -52.7°. The average of

these is close to that of the Pauling-Corey and the Arnott-

Dover φ values but substantially lower than the average assigned

by Barlow and Thornton from the X-ray crystallographic protein

database.

1

Protection Factors PF_is for Ala HN functions of the

Peptide Series 2 and 3

Protection factors PF_i’s measured for the series 2 Ala_n

peptides allow a residue-by-residue assignment of helicity, and

this quantitative energetic characterization complements the

NMR structural assignments. PF_i’s provide the definitive test

â

of the efficiency of helix stabilization by the AspHel N-cap

and beta C-cap, and for representative members of series 2 and

3 they allow calculation of length-dependent FH values. For

series 2, they also allow calculation of site helicities FH_i.

A PF_idefines the relative tendency of a backbone amide NH_i

at site i of a peptide or protein sequence to undergo an NH_if

ND_iexchange reaction in D₂O. An amide NH_ithat is part of

the H-bonded structure of completely helical peptide exchanges

very slowly.⁴¹The exchange rate for an NH at site i of a frayed

helix reflects the presence of conformers in which the amide

NH_iis nonhelical and solvent exposed. A PF_iis defined, eq 2,

Figure 7. Assignment of dominant R-helical structure to the ¹³C- and ¹⁵N-

labeled peptides Ala₈,1, and Ala₁₂, 2. (a) Results of 2D H(N)CO NMR

experiments in water at 10 °C, pH > 4.5, applied to Ala₈. Cross-peaks

(magenta) result from an experiment with dephasing time optimized for

PF_i) k_2NHStd/k_2PeptideNHi

(2)

as the ratio of two rate constants, k_2PeptideNHi, the exchange rate

constant for an NH at site i of a partially helical peptide, and

1

correlation of chemical shifts for a site i ¹³CdO with the site i + 1 HN.

Cross-peaks (green) result from a dephasing time optimized for detection

of ^3hJ_NC′scalar coupling. These demonstrate hydrogen bonds linking residues

1 and 5, 2 and 6, and through a confluent pair of cross-peaks, 3 and 7, 4

and 8: an R-helix. (b) Results of application of the experiments of Figure

7a to the Ala₁₂peptide with six labeled C-terminal residues. The green

cross-peaks correspond to the expected R-helical hydrogen bonds that link

(34) Besley, N. A.; Hirst, J. D. J. Am. Chem. Soc. 1999, 121, 9636-9644.

(35) Pauling, L.; Corey, R. B.; Branson, H. R. Proc. Natl. Acad. Sci. U.S.A.

1951, 37, 205-211.

(36) Barlow, D. J.; Thornton, J. M. J. Mol. Biol. 1988, 201, 601-619.

(37) Weisemann, R.; Ru¨terjans, H.; Schwalbe, H.; Schleucher, J.; Bermel, W.;

Griesinger, C. J. Biomol. NMR 1994, 4, 231-240.

residues 7 with 11 and 8 with 12. Gold crosses mark the expected positions

3h

(38) Vuister, G. W.; Bax, A. J. Am. Chem. Soc. 1993, 115, 7772-7777.

(39) Pardi, A.; Billeter, M.; Wu¨thrich, K. J. Mol. Biol. 1984, 180, 741-751.

(40) Ludvigsen, S.; Andersen, K. V.; Poulsen, F. M. J. Mol. Biol. 1991, 217,

731-736.

for 3₁₀

J

coupling; these cross-peaks are not detected. (c) Top

NC′

spectrum: a slice of an optimized Figure 7b spectrum at the residue 8 ¹³Cd

O resonance (180.45 ppm). Bottom spectrum: a corresponding slice for

residue 7 (180.61 ppm). Gold arrows identify sites of undetected 3₁₀cross-

peaks.

(41) Englander, S. W.; Kallenbach, N. R. Q. ReV. Biophys. 1983, 16, 521-

655.

9

1696 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

k

_2NHStd, the exchange rate constant for solvent-exposed NH in

a simple, analogous model peptide. The atom fraction of the

solvent-exposed NH proton is calculated as 1/PF_i, and eq 3

FH_NHi) 1 - 1/PF_i

(3)

defines the fraction of the NH_ithat is hydrogen bonded within

a helical structure, which is FH_NHi, a measure of local helicity.

Although the ¹³CdO and ¹³CR chemical shifts of an ith amino

acid residue can also be used to calculate site helicity FH_i,²⁷

FH_NHiis more valuable for study of highly helical peptides.

The chemical shift reflects the fraction of the conformer

population that is helical at a ¹³C site, but a PF_iis proportional

to the fraction of nonhelical conformers, which varies by a factor

of 10 if FH_iincreases from 0.90 to 0.99. PF_i’s can be used to

draw fine distinctions within a series of highly helical sites.⁴²

Practical features of the measurement of PF_i’s have been

reported by Englander et al.; these include quantitative effects

of pertinent experimental variables and a database of reference

rate constants k_2NHStdneeded to calculate PF_i’s.¹¹At a given

pH the overall exchange rate constant is a sum of acid- and

base-catalyzed terms, but for normal peptides studied at pH

values greater than 3.5, the base-catalyzed mechanism dominates

and the second-order rate constant depends on concentrations

of hydroxide and substrate as well as temperature and the

hydrophobic bulk and charge of the two amino acids that flank

the exchanging amide NH_i. Corrections for these effects have

been reported.¹¹

Figure 8. Average values of protection factors PF_i’s measured in D₂O at

2 °C pH > 4.5 for Ala_nregions of series 2 peptides with n > 8. Variations

within one SD unit are shown as red and blue bars; the green region

corresponds to the low average and large range of uncertainty for PF_n+1

(see text for details).

thus corresponds to the relative abundance of conformations

that are helical at sites (i - 1), (i - 2), and (i - 3).

Figure 1c depicts the sequence of hydrogen bonds that can

be formed by the amide residues of a representative Ala_nregion

of series 2 and 3 peptides. Three of these are absent for an Ala_n

region that is N-capped by an acetyl and C-capped by an NH₂.

The green hydrogen bonds are common to both regions and

consists of bonds formed between pairs of Ala amides or that

are formed between a single Ala amide NH at site 4 and N-acyl

carbonyl oxygen or the (n - 3) amide carbonyl and the

C-terminal NH. The first three NH sites of a simple Ac-Ala_n-

NH₂are solvent exposed, their PF_ivalues at these sites equal

1.0, and their corresponding FH_NHiequal 0. As assigned from

experimental PF_i, helical stability for a peptide of length n is

defined by (n - 2) nonzero FH_NHi’s at sites i ) 4 through (n +

1).

The PF_imeasurements for Ala NH_i’s of series 2 and 3

peptides were carried out in D₂O at 2 °C. Rates were measured

at pH values above 4.5 and were followed for at least three

half-lives by analyzing both peak heights and curve-fitted areas

1

for the HN NMR resonances. Rate constants for series 2 and

3 peptides were in reasonable agreement.

We have proposed the blue hydrogen bonds of Figure 1c as

linking NH residues of the first three residues of the helix to

oxygens of the AspHel N-cap. The PF₁, PF₂, and PF₃values

As noted earlier, the presence of multiple Inp residues within

the sequences of series 3 peptides results in substantial broaden-

ing and overlap of the ¹HN resonances. The normal resonance

widths observed for series 2 peptides allowed PF_iand FH_NHi

site assignments to the four NHs at the N-terminus of each Ala_n

sequence and to the corresponding four NHs at the C-terminus.

Figure 8 plots averages with standard deviations of these PF_i’s

at each Ala NH site. The central region of the graph plots

averages of PF_i’s for the 20 nearly coincident resonances of

the Ala_ncores of series 2 peptides. These averages exceed 100,

and with their likely error limits, they define limiting core FH_NHi

values as lying within the range 0.990 to 0.994. Individual

resonances usually could not be resolved for series 3 peptides,

but the time dependences of the collective integrated ¹HN

resonance intensities were compared for the two series and were

found to agree within measurement error.

â

reported in Figure 8 demonstrate their presence and their helix-

initiating efficiency. These PF_icorrespond to FH_NHivalues of

0.98 to 0.99. The value of FH_NH1solely reflects interactions of

the first peptide NH with the N-cap. Formation of an intramo-

lecular hydrogen bond by the NH of Ala-1 requires adoption

â

of the helix-initiating (te) conformation by AspHel, and the

magnitude of FH_NH1corresponds to a t/c value equal to or

greater than 50, in accord with the estimates cited in earlier

sections. The values for FH_NH2and FH_NH3reflect the adoption

of helical φ, ψ angles by residues 1 and 2, a structural

precondition for formation of hydrogen bonds between their

NHs and carbonyl oxygens of the pair of tertiary amides within

the Hel function. Finally, FH_NH4reflects the energetics of

formation of the R-helical loop that defines helical φ, ψ angles

for the first three residues of the Ala_nsequence. The small

What structural properties of a peptide helix are reflected by

an FH_NHivalue? Each FH_NHicorresponds to the overall mole

fraction of peptide conformers for which the amide NH at site

i forms a helical H-bond. This bond can be formed, only if each

of the sets of φ and ψ dihedral angles at R-carbons allow the

three preceding residues to form helical H-bonds. An FH_NHi

standard deviations and similar values seen for FH_NH1, FH_NH2

,

FH_NH3, and FH_NH4suggest that no structural mismatch occurs

at the junction between the N-terminus of the peptide helix and

â

the preorganized AspHel N-cap. They confirm the presence

of the H-bonds of Figures 1c and 4b. A total of (n + 1) nonzero

FH_NHi’s characterize sites i ) 1 through (n + 1) of series 2 and

3 peptides.

The remaining (n - 2) PF_i’s of Figure 8 characterize the

helicity of the Ala_nregion itself, and those defined at sites 4

(42) This point is demonstrated by trios of values for (1) PF_iin the range of 2

to 100, (2) FH_NHicalculated from eq 4, and (3) the percent error in FH_NHi

under the assumption of a 10% error in PF_i. For example: 2, 0.5, 24%; 5,

0.80, 7%; 10, 0.90, 2%; 20, 0.95, 1%; 50, 0.98, 0.4%; 100, 0.99, 0.2%. PF_i

values of 50 ( 5 thus allow assignment of FH_NHiwith greater than two-

decimal-place precision.

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1697

A R T I C L E S

Heitmann et al.

Table 1. Length-Dependent FH Values for Series 2 Ala_nPeptides

in Water at 2 °C

through n reflect NH f ND exchange properties of Ala NH

residues. An additional PF_iat site (n + 1) reflects the relative

exchange rate of the R-amide NH of the C-capping function

beta. It monitors local helicity for the three contiguous residues

at the Ala_nC-terminus. Owing to the rapid exchange of this

NH at a pH > 4.5, accurate time-dependent rates could not be

assigned, but upper and lower bounds for PF_(n+1)were estimated

from the pH dependence of the NH resonance broadening in

H₂O within a pH region where intermediate line broadening

due to proton exchange is observed. Calibration through

measurement of an exchange rate for an Ala NH in a short

model peptide showed consistency with PF_ivalues measured

at internal Ala_nsites. The resulting helicity limits are 0.1 <

FH_NH(n+1)< 0.7.

c

n

calculated FH^a

extrapolated FH^b

extrapolated n

8

0.862, 0.944

0.90 ( 0.04

0.91 ( 0.04

0.92 ( 0.03

0.93 ( 0.03

0.94 ( 0.03

0.95 ( 0.02

0.96 ( 0.02

7.2 ( 0.3

8.2 ( 0.4

9

10

11

12

14

16

18

20

22

24

0.888, 0.954

9.2 ( 0.3

10.1 ( 0.3

11.2 ( 0.4

13.2 ( 0.4

15.2 ( 0.3

17.1 ( 0.4

19.2 ( 0.4

21.1 ( 0.4

23.0 ( 0.5

0.905, 0.961

0.918, 0.966

0.927, 0.970

^aThe two series of FH correspond respectively to FH_n) 0.1, combined

the lower limits for other FH_i’s for the series 2 peptides, and to FH_n) 0.7,

combined with the upper limits for other FH_i’s. ^bValues calculated from

values in the first two columns, as described in the text and Appendix section

A monotonic decrease with length is seen for PF_n-3, PF_n-2

,

PF_n-1, and PF_nvalues. A plot of PF_ivs site for a helical peptide

that lacks strong cap stabilization has an inverse parabolic

appearance with a maximal value at site i ) (n/2 + 2) and values

at the terminal sites 4 and (n + 1) that can fall below 1.1.^8,11

The C-terminal length decrease seen in Figure 8 is much less

dramatic. The PF_nfor the C-terminal Ala NH is slightly below

20, corresponding to an FH_NHnof 0.94. This value is consistent

with substantial helical stabilization by the positively charged

C-terminal beta, consistent with the positioning of its ammonium

ion at the C-terminus, as seen in Figure 4a.

c

of the Supporting Information. n ) nFH.

slightly lower value, 0.987, which, from eq 5, can be assigned

to FH₁. Application of eq 6 yields all but one of the remaining

assignments: FH_NH9) FH₈) 0.988; FH_NH10) FH₉) 0.986;

FH_NH11) FH₁₀) FH_NH12) FH₁₁) 0.955. Generalized to the

series 2 Ala_npeptides for n > 8, this analysis yields FH_i

assignments for Ala sites of series 2 peptides. Setting FH₁₂

)

FH_NH13yields the previously defined lower and upper limits of

0.1 and 0.7.

What is the relationship between the set of FH_NHiassigned

for the series 2 peptides and the corresponding site helicities

FH_i? An FH_iis defined as the atom fraction of helical R-carbons

at site i, and an Ala_npeptide has one FH_ifor each Ala R-carbon.

Their average is the peptide fractional helicity, FH. Each of the

(n - 2) FH_NHireflects local three-residue helicity at sites (i -

1), (i - 2), and (i - 3), and as seen in eq 4,

For a series 2 Ala_npeptide and a given value of n, the average

of all FH_ivalues is its overall fractional helicity FH, and the

sum of all FH_ivalues is the average helical length n , eq 7.

(1/n)ΣFH_i≡ FH; n FH ) ΣFH_i) n

(7)

By averaging the lower and upper bounds of error limits for

each FH_ivalue, lower and upper bounds for FH were calculated,

and these are listed in the first and second columns of Table 1.

Consistent with this analysis, interpolated and extrapolated

length-dependent values are calculated by deleting or adding

core values to the calculation, as described in the Supporting

Information. The averages and ranges of these are given in the

third column. Multiplication of each value by n yields values

and ranges for the average helical lengths n ; clearly n z n

-1.

The CD analysis of the following section relies on series 3

data, and an essential issue is a confirmation that the FH values

of Table 1, which were derived from series 2 data, are relevant

to the CD analysis. This confirmation can be obtained by

applying the same FH criterion to members of both data sets.

Weighted averages of PF values derived from time-dependent

integrations of the entire Ala_nNH resonance region for series

2 peptides, n ) 14 and 16, yield a mean FH of 0.94 ( 0.02.

Similar averages of PF values were conducted for series 3

peptides and converted to FH values to yield, for n ) 18, 0.95

( 0.02, and, for n ) 22, 0.955 ( 0.02. These are within the

error limits of the corresponding values seen in the third column

of the table. This finding justifies application of its extrapolated

FH and n value to CD data for both peptide 2 and peptide 3

series.

FH_NHie (FH_i-3+ FH_i-2+ FH_i-1)/3

(4)

the value of FH_NHisets a lower bound for the average of the

43

values of FH_i-1, FH_i-2, and FH_i-3

.

If the value of a PF_i

approaches or exceeds 100, the lower bound can be replaced to

a good approximation by an equality. For example, if FH_NH4

closely approaches 1.0, so must FH₁, FH₂, and FH₃. If, in

addition, the contiguous FH_NH5z 1.0, then FH₄z 1.0. This

pair of FH_NHidefines the values of four FH_i’s. Generalizing

this result to a region, one sees that if the values for FH_NHifor

j contiguous NHs are close approximations to 1.0, it follows

that j + 2 of the associated FH_iare also approximated by 1.0.

The sets of n FH_iand (n - 2) FH_NHithus contain equivalent

conformational information. As shown in the Supporting

Information, the inequalities of eqs 5 and 6

For FH_NHi> 0.9, and i < n/2, FH_i-3z FH_NHi

For FH_NHi> 0.9, and i > n/2, FH_i-1z FH_NHi

(5)

(6)

allow assignment of FH_ivalues for sites at or near the ends of

an Ala_nsequence. The case of an Ala₁₂peptide is representative.

At sites 5 through 8, the FH_NHivalues are 0.992, implying that

FH_ig 0.99 at sites 2 through 7. At site 4, FH_NHiassumes a

The results of all NMR analyses can be summarized. (1) The

helical region is confined to the Ala_nsequences, which form

R-helices with φ angles consistent with polyalanine fiber

(43) As detailed previously⁸(also see Supporting Information) the conformations

of the manifold for FH_NHiconsist of the logical intersection of sets of

conformations that define the values of FH_i-1, FH_i-2, and FH_i-3. For highly

helical peptides and for either small or large values of i, this intersection

set is closely approximated by the conformations of the manifold associated

with the smallest FH_i.

1

diffraction data. (2) From NOEs and modeling evidence, H-

¹H contacts detected between the N-cap and the C-cap Ala_n

9

1698 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

sequence occur, respectively, with the first and last three

sequence residues, as depicted in Figures 1c and 4. (3) For n >

8, both chemical shifts and PF measurements identify core

regions of length n - 8, consisting of Ala residues with

essentially invariant NMR properties. (4) For the pairs of four

residues at the cap-Ala_njunctions, both chemical shifts and PF_i

values depend on the site i but are independent of the overall

n. (5) The FH_ivalues for residues in the core regions exceeds

0.99. (6) The mean Ala_nFH at 2 °C in water lies in the range

0.90 to 0.96.

These data are consistent with the principle advanced in an

earlier section, that the structural effects of the helix-stabilizing

N- and C-caps of the Ala_npeptides of series 2 and 3 should

perturb helix structure locally, but not globally. Deviations from

core helical properties are confined to the eight Ala residues at

the helix ends. This is the necessary condition for applying 2

and 3 Ala_npeptides n > 8 as standards for CD calibration.

A more recent role for CD-derived FH values requires a

higher accuracy. Predictive algorithms, constructed to assign

peptide helicity from amino acid structure and sequence, are

often based on FH values calculated from [θ]_222,

data

Exp

measured for host-guest substitutions in alanine-rich peptides.^13a,b

We have recently argued that spaced, solubilized Ala_npeptides,

Figure 1a, are the natural hosts for host-guest helicity studies,⁵

and as a preliminary to host-guest studies, we have presented

CD and t/c evidence characterizing helical propensities for the

Ala residue.^5b.cWe have also noted that literature values for

[θ]_λ,∞fail as satisfactory models for the CD properties of

(Ala₄Lys)_npeptides and polyalanines, which usually exhibit

exceptionally large ratios of [θ]₂₂₂to [θ]₂₀₈as well as curved

parametric plots of temperature-dependent [θ]₂₂₂vs [θ]₂₀₈

data.^12bUsing the calibration peptides of this study, we now

clarify these issues.

[θ_{Molar λ,Ala,n}

[θ_{Molar λ,n,Exp}

]

) n[θ]_λ,n) n[θ]_λ,∞- X[θ]_λ,∞

(9)

Assignment of Core Per Residue Molar Ellipticities

[θ]_λ,∞ from Experimental Molar Ellipticites [θ_Molar

]

for

λ,n

]

) [θ]_λ,∞(n - k) + [θ]_λ,∞(k - X)

(9′)

Series 3, n ) 9 through 24, and Series 2, n ) 10

]

) [θ_{Molar λ,AlaCore,n}

]

+ [θ_Molar]_λ,SS-Caps)

through 16; Calculation of the Limiting Polyalanine CD

Spectrum in Water at 2 °C. Calculation of the Limiting

Polyalanine CD Spectrum in Water at 2 °C. Assignment

of the Length Parameter X

[θ]_λ,∞(n - k) + [θ_{Molar λ,SS-Caps}

]

(10)

As a preliminary to data analysis, we rewrite eq 1 as its molar

equivalent, eq 9. With no loss of generality, eq 9 can be rewritten

as 9′, which introduces a parameter k that corresponds to the

number of alanine residues of Ala_nwith ellipticity contributions

that are perturbed from core values by capping or other end

effects. The NMR data of preceding sections suggest an

assignment of k ) 8, subject to CD validation.

Two assumptions underlie application of eqs 1 and 9′ and

their equivalent, eq 10, to the calibration series.⁴⁸First, for any

series 2 or 3 peptide that contains an Ala_ncore with convergent

The use of eq 8 and eq 1 to calculate FH values for peptides

and local sequences of proteins from experimental CD residue

ellipticities [θ]_222,n,Exphas a long history.¹⁶Owing to the strength

of the limiting helical ellipticity [θ]_222,nrelative to that of other

conformations, the rigorous form of eq 8 is commonly ap-

proximated as a simple ratio of experimental to limiting residue

ellipticities.

FH ) ([θ]_222,n,Exp- [θ]_222,RC)/ ([θ]_222,n- [θ]_222,RC) z

[θ]_222,n,Exp/[θ]_222,n(8)

properties, the value of [θ_{Molar λ,n,Exp}

proximated as a sum of two regional molar ellipticities, [θ_Molar

_,coreand [θ_{Molar λ,SS-Caps}; second, the former, which is the core

]

can be accurately ap-

]

[θ]_222,n) [θ]_222,∞(1 - X/n)

(1)

λ

]

molar ellipticity, can be written as [θ]_λ,∞(n - k), where (n - k)

is the core length, and [θ]_λ,∞is the constant incremental increase

in peptide molar ellipticity that results if a core region length is

increased by one residue.

Recent developments have increased the accuracy with which

protein conformations can be assigned from CD data for proteins

of known native structure, and more than thirty years of

deconvolution of CD spectra for highly helical proteins of

known native structure^44,45have provided reasonably satisfactory

assignments for the empirically defined parameters X and [θ]_λ,∞

of eq 1. However, errors of fit to helices of the protein database

remain large.³⁴Extrapolations of solvent dependencies for the

CD properties of helical alanine-rich peptides of short-to-

medium length have also yielded values for X and [θ]_λ,∞that

are in approximate agreement with protein assignments,⁴⁶

although the assumptions underlying these extrapolations have

been recently questioned.¹²FH values calculated in this manner

are used to rank the “ballpark” helicities of heteropeptides and

to monitor temperature or denaturant-induced unfolding of

helical proteins.⁴⁷

Figure 9 shows λ-plots of [θ_Molar] _λ,n,Expfor five representative

lengths of the series 3 peptides for which n > 8. The evident

strong length dependence reflects dominance of ellipticities

attributable to the Ala_ncores over the capping effects, which

include ellipticity contributions by the first and last four residues

of the Ala_nsequence. In the previous section, PF_imeasurements

for core regions were observed to be length independent, with

limiting FH_ivalues of at least 0.99. This result implies that the

per residue increase in [θ_Molar

]

does not reflect a length-

λ,n,Exp

dependent increase in FH_i. It must be attributed to the

incremental effect of adding a new, fully helical Ala residue.

Is [θ_Molar]_λ,SS-Capsin fact length-independent, consistent with

the length independence of ¹H chemical shifts and PF_i’s (Figure

8), and as required by eq 10? A first answer is provided by

calculated regression intercepts for n ) 8, which can be

(44) (a) Greenfield, N. J. Anal. Biochem. 1996, 235, 1-10. (b) Sreerama, N.;

Venyaminov, S. Y. Woody, R. W. Anal. Biochem. 2000, 287, 243-251.

(45) (a) Woody, R. W. J. Polym. Sci. Macromol. ReV. 1977, 12, 181-320. (b)

Manning, M. C.; Woody, R. W. Biopolymers 1991, 31, 569-586.

(46) (a) Gans, P. J.; Lyu, P. C.; Manning M. C.; Woody, R. W.; Kallenbach, N.

R. Biopolymers 1991, 31, 1605-1614. (b) Scholtz, J. M.; Qian, H.; York,

E. S.; Stewart, J. M.; Baldwin, R. L. Biopolymers 1991, 31, 1463-1470.

(c) Luo, P.; Baldwin, R. L. Biochemistry 1997, 36, 8413-8421.

(47) Peterson, R. W.; Nicholson, E. M.; Thapar, R.; Klevit, R. E.; Scholtz, J.

M. J. Mol. Biol. 1999, 286, 1609-1619.

(48) For a detailed discussion of this point, see the Supporting Information.

The equivalence of eqs 1, 9′, and 10 follows directly from: X ≡ k -

[θ_Molar]_λ,SS-Caps/[θ]_λ,∞; the ratio of this assignment scales the molar ellipticity

[θ_{Molar λ,SS-Caps}as a length by dividing it by the core per residue molar

]

ellipticity, [θ]_λ,∞; X is thus the difference between the actual length k and

this scaled length.

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1699

A R T I C L E S

Heitmann et al.

Figure 9. Wavelength plots of [θ_Molar

]

for series 3 Ala_npeptides in

λ,n,Exp

Figure 11. Extrapolated CD spectrum [θ]_λ,∞of a completely helical Ala_n

peptide in water at 2 °C. The red dots correspond to the slopes calculated

water pH > 4.5, 2 °C. Units: deg cm²dmol^-1. Spectra increase in intensity

through the series n ) 9 (green), 12, 16, 20, 24 (blue-green).

from the linear length correlations of [θ_Molar

]

that appear in Figure 12.

λ,n

Units: deg cm²dmol^-1residue^-1. Except for the two regions within which

[θ]_λ,∞approaches 0, its least squares error is ca. 2% of its value.

CD spectrum of a fully helical alanine residue within the core

region of a polyalanine helix, measured in water at 2 °C. The

spectrum is qualitatively representative of benchmark literature

spectra for limiting R-helices, but it is distinguished by the

intensity of [θ]_222,∞and by the ratio [θ]_222,∞/[θ]_208,∞) 1.30,

larger than most literature values but consistent with our

previous reports for alanine-rich peptides.^12b

To calculate both X and [θ]_λ,∞, we corrected series 3

[θ_{Molar 222,n}

[θ_{Molar 222,0}values and dividing the resulting differences by the

]

data for capping contributions by subtracting

]

series of FH values listed in Table 1. In the normal process for

calculating the FH of an n-residue peptide, eq 8, the experi-

mental [θ]_222,n,Expvalue is divided by [θ_Molar]_222,n, calculated by

Figure 10. Length-dependent linear regressions at four wavelengths of

[θ_Molar

]

for series 3 Ala_npeptides, n > 8. Units: deg cm²dmol^-1. Data

λ,n

from the spectra of Figure 9 appear as correspondingly colored dots.

Extrapolated values for k ) 8 appear at the intersection of the red lines

with the vertical axis.

applying literature values of X and [θ]_222,∞to eq 1. This process

is reversed to calibrate X and [θ]_222,∞from corrected molar

ellipticities. Three linear length regressions were carried out

using the low error limits, the mean values, and the high error

limits of FH. Resulting values for [θ]_222,∞are -61 200 (SD

1300), -60 500 (SD 1250), and -59 800 (SD 1200). Corre-

sponding values for X are 2.6 ( 0.3, 2.8 ( 0.3, and 2.9 ( 0.3.

Similar results are obtained if both series 2 and series 3 peptides

are included.⁴⁹

compared with the experimental values for [θ_Molar]_λ,8,Exp. At all

wavelengths, these values agree within measurement errors. Data

at 201 nm provide a second answer. At this wavelength, the

contribution to [θ_{Molar λ,n,Exp}

consistent with the isoellipsoidal point of Figure 9. Equation

]

from the Ala_nhelix vanishes,

10 is reduced to its second term. The values of [θ_{Molar 201,n,Exp}

]

in units of 10³deg cm²dm^-1for the 10 series members are n

) 9, -254.3; 10, -255.6; 11, -250.6; 12, -254.6; 14, -253.6;

16, -265.2; 18, -242.5; 20, -245.0; 22, -233.0; 24, -277.2.

The ellipticity-length correlation coefficient is -0.002. There

is no detectable length dependence of the capping contribution.

Values of [θ]_208,∞are also relevant to FH calculations. Linear

length regressions of the corrected [θ_Molar]_208,n,Expdata set yield

values for [θ]_208,∞of -46 000 deg cm²dmol^-1with X in the

range 4.3-5.1. Notably, this [θ]_208,∞lies close to the range of

literature values.

At constant λ, from eq 10, [θ_Molar

]

values should

λ,n,Exp

correlate strictly linearly with n. The quality of this correlation

at four wavelengths is seen in Figure 10. A quantitative test of

linearity is provided by linear length regressions applied to

Discussion

How general are the results of this study, which has been

directed at clarifying problems with assigning NMR parameters

and CD-based FH values to polyalanines and by analogy to

alanine-rich peptides? The value of [θ]_222,∞, water 2 °C, assigned

in this report, is 40% more intense than the upper limit of current

literature values,^34,46which fall in the range -37 × 10³to -44

× 10³deg cm²dmol^-1residue^-1. For typical helical peptide

fragments derived from globular proteins, this range remains

appropriate, but it is almost certainly underestimates [θ]_222,∞

for that class of helically disposed peptides that exhibit a ratio

[θ_{Molar λ,n,Exp}data sets from which the shorter Ala_nlengths are

]

deleted. If core convergence is valid, the assigned slopes must

be invariant. At 222 nm, the linear regression of the full data

set yields a slope: -59 600 (SD 1300), and an intercept:

202 000 (SD 20 700); if the first two data points are deleted

(data set: 11 e n e 24), the slope is -59 300 (SD 1700); further

restriction (data set: 14 e n e 24) yields a slope of -58 800

(SD 3100). Within standard error, these assignments are

identical, and no deviations from linearity are detected at either

low or high length limits.

(49) Least squares linear length regressions of combined, corrected series 2 and

Slopes for these regressions can be equated with [θ]_λ,∞, and

these values are plotted in Figure 11. This is the first extrapolated

series 3 [θ_Molar

20% decrease in X.

]

data resulted in a ca. 2% decrease in [θ]_222,∞and a

222,n

9

1700 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

Figure 13. Lifson-Roig modeled⁸FH values for a 16-residue homopeptide

assigned a helical propensity w of 1.2. Blue curve: site-dependent FH values

calculated for N- and C-terminal capping parameters of 1.0. Green curve:

results of increasing capping parameters to 7.0. Red curve: N-capping

parameter of 200; C-capping parameter of 7.

Figure 12. Green region graphs the length dependence of [θ]_222,n, eq 1,

calculated from [θ]_222,∞) -60 500 (SD 1250) deg cm²dm^-1residue^-1

and X ) 2.8 ( 0.3 residues. Values of the 10 experimental ([θ_Molar

]

-

222,n

[θ_Molar]_222,0)/FH values used to assign X and [θ]_222,∞are plotted (red); for

comparison, two corresponding values calculated from [θ_Molar]_222,n, n ) 6

and 8 are also shown (violet).

Why have we failed to detect 3₁₀structure for our series 2

N- and C-capped Ala_npeptides? The strong bias toward

R-structure provided by the three preorganized hydrogen bond-

ing sites of the ^âAspHel N-cap is a likely answer. Can the

observed examples of 3₁₀structure seen with peptides derived

from simple amino acids result from the reduced hydrogen

bonding potential of water molecules or other functions available

at helix ends? The presence of only two unsatisfied N-terminal

NH hydrogen bonding valences for the 3₁₀helix may reduce

its sensitivity to these factors.

Lifson-Roig modeling⁸provides insight into the magnitudes

of the helix-inducing capping effects we have observed in this

study. The blue curve of Figure 13 demonstrates typical FH_i

values for a medium-sized helically disposed homopeptide that

lacks stabilizing N- and C-caps. Conformational averaging or

“fraying” is responsible for the marked decrease of FH_ithat is

evident in the end regions.

[θ]₂₂₂/[θ]₂₀₈greater than 1.2 and curved parametric [θ]₂₂₂vs

[θ]₂₀₈plots.^12bThese include polyalanines and alanine-rich

peptides. For these, our new [θ]_222,∞value of -60 500 ( 1300

is the appropriate choice.

Figure 12 shows the calibration curve that results if the

parameters derived in the preceding section are applied to eq

1. The large uncertainties in X generate correspondingly large

errors in [θ]_222,nand FH. For a peptide of 24 residues the error

in [θ]_222,24is (4%, but if the length is halved the error increases

to (7%. Is there a need to calibrate helicity for short peptides?

Peptides in the length range of 5 to 12 residues have been

reported to exhibit weak CD spectra with helical features.⁵⁰

Recently reported examples include short, lanthanide cation-

stabilized helical polyalanines.⁵¹Figure 12 provides needed “ball

park” estimates of limiting [θ]_222,nvalues for short peptides.

Can one improve the precision of X assignments for water-

solubilized peptides? Unlike [θ]_λ,∞, which is defined largely by

the CD properties of medium to long helical peptides, the X

The green curve results from a recalculation that introduces

stabilizing N- and C-caping effects. A marked increase in FH_i

is seen which is largest at the ends. A leveling is evident in the

central region. Both effects are attributable to an increase in

the mole fractions of substantially helical conformations. The

red curve shows the effect of retaining a moderately stabilizing

C-cap and introducing a highly stabilizing N-cap. The asym-

metric site dependence of FH_inow shows little or no hint of

fraying at the N-terminus. We believe the parameters used in

calculating the red curve are reasonable approximations to the

assignment must be based on experimental [θ_Molar

]

data for

λ,n

short peptides. These data have intrinsically problematic features.

Large assignment errors attend their weak ellipticities, and these

are compounded by length uncertainties.

Since X reflects local effects of N- and C-caps, it must ideally

be tailored to the particular capping structure of each new

peptide series. The value of X is likely to depend on the nature

of the polar atoms or charges that appear within end regions,

which can influence N- and C-terminal conformations. The

contribution of 3₁₀helical conformers to helical peptides of short

to medium length⁵²poses a further ambiguity, since [θ]₂₂₂is

weak for 3₁₀helices,⁵³and variations in the R/3₁₀ratio are poorly

understood.

â

capping properties of AspHel and beta, yet this curve under-

estimates the FH values we have documented for the capped

peptides of this study. A second essential requirement for

achieving FH values close to 1.0 is the presence of an amino

acid with a high helical propensity. We have demonstrated a

substantial length increase of the helical propensity w of alanine

for short to medium-sized polyalanines,^5cand subsequent studies

show that w assumes large values for peptides containing 20 or

more residues.⁵⁴This length dependence contributes to confor-

mational homogeneity and compensates for an expected decrease

in cap efficiency as the helical length is extended.

(50) (a) Zerkowski, J. A.; Powers, E. T.; Kemp, D. S. J. Am. Chem. Soc. 1997,

119, 1153-1154. (b) Gallo, E. A.; Gellman, S. A. J. Am. Chem. Soc. 1994,

116, 11560-11561. (c) Karle, I. L.; Balaram, P. Biochemistry 1990, 29,

6747-6756. (d) Parthasarathy, R.; Chaturvedi, S.; Go. K. Proc. Natl. Acad.

Sci. U.S.A. 1990, 87, 971-875.

(51) (a) Siedlecka, M.; Goch, G.; Ejchart, A.; Sticht, H.; Bierzynski, A. Proc.

Natl. Acad. Sci. U.S.A. 1999, 96, 905-908. (b) Chin, D.-H.; Woody, R.

W.; Rohl, C. A.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,

15416-15421.

(52) (a) Miick, S. M.; Martinez, G. V.; Fiori, W. R.; Todd, A. P.; Millhauser,

G. L. Nature 1992, 359, 763-655. (b) Long, H. W.; Tyko, R. J. Am. Chem.

Soc. 1998, 120, 7039-7048.

(53) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.; Kamphuis, J. J. Am.

Chem. Soc. 1996, 118, 2744-2745.

(54) Kennedy, R. J., Ph.D. Dissertation, MIT, February, 2004.

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1701

A R T I C L E S

Heitmann et al.

Our success with generating a tractable length series of

polyalanines with exceptionally high helicity almost certainly

guarantees extension of the concept to other peptide classes.

Previously studied highly helical alanine-rich heteropeptides are

obvious candidates, but work in several laboratories has also

demonstrated that unnatural amino acids with straight aliphatic

side chains can have helical propensities equivalent to that of

alanine.⁵⁵Provided aggregation can be inhibited by appropriate

solubilization, these are obvious candidates for future study. A

necessary condition for all such studies is proof that the currently

available strongly helix-inducing N- and C-caps can function

as efficiently for more general peptide series as they have for

the polyalanines. A particularly important goal is improving the

efficiency of helix-stabilizing C-caps.

interpretation of subtle conformational features of helical

peptides that currently lie beyond its scope.

Summary

N- and C-capped polyalanines that lack helical stabilization

through tertiary interactions can approximate single fully helical

structures. Two series, with general structures 2 and 3, have

been shown by AUC to be unaggregated in water. NMR

experiments with series 2 peptides have characterized structure

and energetics. Literature-precedented conformations of the

^âAspHel N-cap and the C-cap beta have been shown to interact

and stabilize the first and the last helical loops of the Ala_n

sequences. The fully ionized carboxyl function of the N-cap,

pK_a3.27, is required for maximum helical stability. Analysis

of Ala PF_ivalues and chemical shifts of Ala resonances lead

us to the conclusion that the properties of the capped, solubilized

Ala_npeptides can be classified into N-cap, core, and C-cap

regions. For Ala_npeptides with n > 8, the residue fractional

helicities FH_iof the n - 8 core residues lie at or above 0.99.

The NMR resonances assigned to the central alanine core region

of length n - 8 exhibit convergent helical ¹HN and ¹³C chemical

As tools for molecular engineering, peptides that assume fixed

conformations have seen relatively little application. Given their

conformational homogeneity, the capped Ala_npeptides of this

study may become an important exception. They provide

versatile cylindrical structures with defined lengths that can be

extended within wide limits. Almost certainly these constructs

will find roles as substrates for further NMR characterization

of water-soluble peptide helices that lack stabilization from

tertiary structure. Conformational averaging has hitherto plagued

interpretation of physical data measured for simple helical

peptides in aqueous solvents, but as this study has shown,

structurally relevant databases comprising chemical shifts and

coupling constants can now be measured through isotopic

labeling of peptides similar to those of Ala_nseries 2.

3

shift values. The measured J_HNHRcoupling constants for the

core region of the Ala₁₂peptide allows assignment of the

backbone dihedral angle φ as - 50.5°, and R-helical structure

3h

is proved by detection of

J

scalar couplings. Core length-

NC’

dependent linear regressions carried out on [θ_{Molar λ,n}values

]

measured for series 2 and 3 with n > 8 residues over the

wavelength range of 195 nm e λ e 350 nm yield slopes that

define the ellipticity parameter [θ]_λ,∞. Collectively these define

the first extrapolated CD spectrum of a fully helical alanine

residue within the core region of a polyalanine helix, measured

in water at 2 °C. The value assigned to [θ]_222,∞in this study is

(-60.5 ( 1.3) × 10³deg cm²dm^-1, confirming our previous

approximate assignments.^5,12aThe limiting ellipticity value

[θ]_208,∞assigned in this study at the 208 nm minimum is (-46

( 2) × 10³deg cm²dm^-1, in approximate agreement with

literature values.

The interpretation of CD spectra of helical peptides raises

many unresolved issues. How good is the widely accepted

approximation that side chain substituents do not influence CD

ellipticity? New second-generation calibration standards should

allow this question to be addressed rigorously. A particularly

important issue concerns temperature effects. Although they are

the formal equivalents of the familiar extinction coefficients of

electronic molecular spectroscopy, [θ] values differ in that they

have been reported as exhibiting significant temperature de-

pendence that may reflect conformational changes. Without

helical calibration standards, and without the support of parallel

NMR studies, it is difficult to draw rigorous distinctions between

ellipticity changes that define this effect and those attributable

to helix melting. We have recently reported a pertinent curvature

test, based on a parametric plot of [θ]₂₂₂vs [θ]₂₀₈data, and

have concluded that [θ]₂₂₂values for alanine-rich peptides are

probably exceptionally temperature sensitive.^12bFurther studies

can be envisaged that employ FH-calibrated ellipticity standards

to identify features of amino acid composition and sequence

Experimental Section

Synthesis, Purification, and Characterization of Peptides. Peptides

were synthesized on a 0.02-0.03 mole scale by automated continuous-

flow solid-phase synthesis using a PE Biosystems Pioneer Peptide

Synthesizer with standard 9-fluorenylmethyleneoxy-carbonyl (Fmoc/

HATU) chemistry, and peptides were cleaved from the resin as reported

previously.^5,9Purification was carried out on a Waters 2690 HPLC with

auto-injector and 996 detector using YMC ODS-AQ 200 Å 4.5 × 150

mm²columns. Characterization tables of mass spectrometric data

obtained on a Waters ZMD mass spectrometer and AUC data obtained

on Beckman Optima XI-A centrifuge equipped with a six-channel cell

in an An-60 Ti analytical rotor can be found in the Supporting

Information. The published synthesis⁹for Fmoc-Hel-OH from Cbz-

Hel-OH was modified as follows: hydrogenation on Pd-C was carried

out in EtOH for 24 h yielding 94% of H-Hel-OH, which was

transformed into Fmoc-Hel-OH, 71%, by prior esterification with TMS-

Cl, reaction with Fmoc-Cl, and hydrolysis, following the procedure of

Bolin et al.⁵⁶The N-Fmoc-trans-4-aminocyclohexane carboxylic acid,

Fmoc-Acc-OH, required for synthesis of peptides of series 2 was

prepared by reaction of trans-4-aminocyclohexane carboxylic acid with

Fmoc-Cl in aqueous buffer. Details of preparation and characterization

that correlate with the large temperature dependence of [θ]₂₂₂

.

The immediate goals of such studies are potentially within reach

and work toward a deeper predictive understanding of the factors

that influence ellipticities for helical peptides.

More ambitious goals can also be envisaged. CD spectroscopy

remains the most convenient, efficient, and inexpensive tool for

detecting and quantitating helical structure within polypeptides

and proteins. One can hope that the scope of CD analysis of

helical peptides can be expanded to include assignment and

(55) (a) Lyu, P. C.; Sherman, J. C.; Chen, A.; Kallenbach, N. R. Proc. Natl.

Acad. Sci. U.S.A. 1991, 88, 5317-5320. (b) Moreau, R.; Kemp, D. S.

Unpublished observations.

(56) Bolin, D. R.; Sytwu, I.-I.; Humiec, F.; Meienhofer, J. Int. J. Pept. Protein

Res. 1989, 33, 353-359.

9

1702 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

H₂O-Solubilized, Cap-Stabilized, Helical Polyalanines

A R T I C L E S

of Fmoc-Acc-OH and preparation of Fmoc-Hel-OH can be found in

the Supporting Information.

Resonance intensities were measured for baseline-corrected 1D ¹H

spectra (16384c, sweep width 8992.9 Hz, presaturated for solvent

suppression) using the integration menu of XWINNMR. For the series

2 Ala₈peptide uniformly labeled with ¹³C and ¹⁵N (see below), a 2D

version H(N)CO of a standard HNCO experiment²⁸in water at 8 °C,

pH 4.5 yielded cross-peaks that correlate ¹³CdO chemical shifts for a

site i Ala residues with ¹HN chemical shifts at the site i + 1. In addition

to the cross-peaks expected for the major resonance, this experiment

also showed much weaker cross-peaks for the minor resonances in the

¹³CdO chemical shift range 177.9-178.5 ppm, characteristic of random

coil values. An approximate integration ratio gave values in the range

40-70 for t/c measured with an Ala₈peptide under these conditions.

Studies of Ac^âAspHelAla_nbetaAccLys₂TrpNH₂n even, 8-16;

Peptide Series 2. Assignment of the Ala ¹H HN resonances was carried

out on each member of the series in the pH range 3.45-5.34 from

standard TOCSY (512c*4096c, τ_m) 70 ms) and NOESY⁶¹spectra

(512c*4096c, τ_m) 400 ms) using a sweep width of 7183.9 Hz in both

dimensions and watergate solvent suppression.

pH Assignments. Measurements were made at ambient temperatures

in the pH range 1 to 9 with a Radiometer Copenhagen PHM240 pH

meter equipped with a Cole-Parmer EW-55529-08 glass electrode,

standardized daily with pH 4.0 and 7.0 standard buffers purchased from

VWR Scientific. The rate measurements for amide NH f ND exchange

were conducted in D₂O; the pD meter readings were converted to pH

values that are cited in the manuscript using the standard formula: pH

) pD + 0.4.

CD Experiments. Circular dichroism measurements were obtained

on an Aviv 62DS circular dichroism spectrometer as described

previously.^5,12bThe instrument was calibrated as described in the

operating manual using previously titrated water solutions of sublimed

9-camphorsulfonic acid. Peptide concentrations were determined on a

Cary 300 UV-vis spectrometer utilizing the Trp chromophore of the

peptide, as previously reported.²From the deviations in linear length

regression of [θ]₂₂₂in the region 12 < n < 19 for the Ala_nseries of

Miller et al.,^5bthe precision of [θ] is taken to lie in the range 3% to

5%.

NMR Experiments. All spectra were collected on a Bruker Avance

600 instrument (Karlsruhe, Germany) equipped with four channels and

a pulsed field gradient triple probe with z gradients. Processing and

data analysis were carried out on a SGI O2 workstation using

XWINNMR 3.5 software (Bruker). Unless stated differently, all spectra

except those for the NH-ND exchange experiments were obtained in

H₂O/D₂O (95/5, v/v), 40 mM phosphate buffer, and spectra were

measured with a relaxation delay of 1.8 s or greater. As noted below,

the pH was adjusted as necessary by addition of small amounts of NaOH

or H₃PO₄. DSS was used as an internal reference for ¹H chemical shifts,

and ¹³C and ¹⁵N chemical shifts were referenced indirectly in accord

with IUPAC recommendations.⁵⁷For data processing in the indirect

dimensions, zero filling was applied, and a square sine bell shifted by

π/2 was used for apodization in all dimensions.

E.COSY HNCA and H(N)CO Experiments with Ac^âAspHelAla₈-

betaAccLys₂TrpNH₂and Ac^âAspHelAla₁₂betaAccLys₂TrpNH₂

Containing Uniformly ¹³C and ¹⁵N Labeled Ala Residues. Eight

residues of the Ala₈peptide were doubly labeled, but to minimize

resonance overlap, Ala₁₂peptides were synthesized in which labeling

was confined either to residues 1-6 or to residues 7-12. The ¹³CR

chemical shifts were assigned from a standard HNCA experiment, and

the ³J_HNHRcoupling constants were assigned from an E.COSY HNCA

experiment (2048c*4c*64c). The sweep widths for both experiments

1

were 5387.5, 426, and 1207 Hz respectively for the H, ¹³C, and ¹⁵N

dimensions. The values for the ¹³CR chemical shifts are shown in Figure

7a,b.

For the two labeled Ala₁₂peptides the ¹³CdO chemical shifts were

assigned from a 2D version, H(N)CO, of a standard HNCO experiment²⁸

(1024c*256c) with a sweep width of 5387 and 980 Hz, respectively,

1

for the H and ¹³C dimensions. For the Ala₁₂peptide with the last six

Studies of the (te) and (cs) Conformations of Ac-^âAspHel-Ala₈-

beta-NH₂Peptide 1. A 1D ¹H NMR spectrum was measured followed

by standard TOCSY⁵⁸(512c*4096c, τ_m) 70 ms) and ROESY⁵⁹

(512c*4096c, τ_m) 300 ms) spectra with sweep widths of 6613.76 Hz

in both dimensions, using presaturation for solvent suppression. From

these spectra, all chemical shifts were assigned as reported in the text,

and a listing appears in the Supporting Information. To assign the pH

Ala residues labeled, the nitrogen to carbonyl dephasing time (2T) was

set to 0.024 s, which corresponds to a value of slightly shorter than

1

1/(2*¹J_NCO), J_NCO) 15 Hz, a commonly used value for correlations

of chemical shift values for a residue i, ¹³CdO with residue i +1 ¹HN.

For the hydrogen bonding experiment,³²data were measured at 10 °C

in 40 mM phosphate buffer, pH 4.46, at a sample concentration of

2-4 mM in a Shigemi NMR tube (Tokyo, Japan) on the Bruker

spectrometer, equipped with a ¹H, ¹³C, ¹⁵N triple resonance 5 mm

CryoProbe. A data matrix of 2048c*32c was collected with a sweep

width of 3894 and 203.7 Hz for the respective ¹H and ¹³C dimensions.

The hydrogen bonding spectrum of the C-terminally labeled peptide,

Figure 7b, was measured with 2T ) 0.1316 and 512 scans, which allows

detection of ^3hJ_NC’scalar couplings.³²Similar measurements were carried

out on the labeled Ala₈peptide and the N-terminally labeled Ala₁₂

peptide, Figure 7a. The spectrum of Figure 7c was measured with 2T

) 0.1303 and 1024 scans, and data from this experiment were used to

calculate signal-to-noise values.

1

dependences of resonances, a series of pH-dependent 1D H spectra

(16384c, sweep width 8992.0 Hz, pH range 1.3-8) were measured for

2

a sample of peptide 1 containing uniformly H-labeled Ala residues.

As reported in the text, significant pH dependences were only observed

for the chemical shifts of three NH resonances.

A second, minor conformation could be detected in some spectra

that showed a distinctive chemical shift of the C-12 resonance, linked

by a cross-peak with a 9-H Hel resonance; these are signature features

of the previously characterized (cs) state of a Hel-peptide conjugate.²⁰

The eight Ala NH resonances of the minor conformation were identified

by their TOCSY signatures; they appeared downfield of resonances

for the major conformation, implying weaker helicity. The resonance

intensities of resonances associated with the (cs) conformation increase

with an increase in temperature and decrease with an increase in pH

or the length of Ala_n. At pH values below 3.8 and for the Ala-deuterated

peptide, the (cs) state is sufficiently abundant to allow t/c to be estimated

as a ratio of intensities of the Hel 12-H (t) and (c) proton resonances.⁶⁰

At pH 3.8, a value of 6 was observed at 320 K, which increased nearly

linearly to a value near 12 as the temperature was decreased to 280 K.

Protection Factor Measurements on Peptides of Series 2 and 3.

A PF_ivalue for base-catalyzed amide NH f ND exchange in D₂O is

the ratio of the second-order rate constant k_2NHStdmeasured for a model

unstructured amide NH and the analogous rate constant k_2PeptideNHi

measured under the same conditions for a backbone NH at site i of a

test alanine peptide. Values reported by Bai et al.¹¹for pH and

temperature dependence of k_2NHStdwere used in this study and

independently confirmed.⁶²Amide proton-deuterium exchange experi-

ments were conducted at 2 °C in D₂O (20 mM phosphate buffer)

containing 1-2 mM peptide samples; the pD measurements for these

experiments were converted to the reported pH values with the standard

equation: pH ) pD + 0.4. An optimal pH for NH f ND exchange

(57) Markley, J. L.; Bax. A.; Arata, Y, Hilbers, C. W.; Kaptein, R.; Sykes, B.

D.; Wright, P. E.; Wu¨thrich, K. Pure Appl. Chem. 1998, 70, 117-142.

(58) (a) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360. (b)

Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521-528.

(59) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 207-213.

(60) Kemp, D. S.; Curran, T. P.; Boyd, J. G.; Allen, T. J. J. Org. Chem. 1991,

56, 6672-6682.

(61) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys. 1979,

71, 4546-4553.

(62) Kennedy, R. J.; Kemp, D. S. Unpublished observations.

9

J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1703

A R T I C L E S

Heitmann et al.

for all alanine NH resonances was found to lie in the range 4.5-4.8,

which ensured that the ^âAspHel helix-inducing cap was maximally

effective and allowed convenient measurement of the slow exchanges

observed for the core alanine NH functions. Time-dependent peak

heights and curve-fitted resonance areas for ¹HN NMR resonances were

carried out for both series 2 and series 3 peptides and found to be in

reasonable agreement; satisfactory fits to pseudo-first-order behavior

were observed over at least three half-lives. Curve fitting was performed

using the spreadsheet and graphing software package Origin. The NMR

spectra were fit with Lorentzian peaks through the Fit Multi-Peaks

option from the Analysis menu. Each identifiable peak was fitted with

a single Lorentzian peak; thus, for the longer peptides, the core alanine

residues were fitted with a single peak. The R²values for the fits were

typically greater than 0.99; the lowest value observed was 0.96. All

fitted peaks had appropriate line widths for amide protons. Owing to

its rapid NH f ND exchange at a pH > 4.5, upper and lower limits

for the PF_n+1value for the NH of beta were assigned by estimating for

an Ala₈peptide the relative pH dependence of the NH resonance

broadening in H₂O in the pH region where intermediate line broadening

due to proton exchange is observed. These were compared with values

for a beta-containing random coil model to estimate likely upper and

lower bounds for PF_n+1.

⁶³Calibration with Ala NHs showed satisfactory

consistency with PF values measured above.

Acknowledgment. Financial support was provided by NSF

Grant CHE-0131250 (G.E.J. and B.H.), by NIH Grant GM13453

(S.M.W. and R.J.K.), and by Pfizer Research. We also thank

the NIH for funding through Grant 1S10RR13886-01 that

allowed purchase of the Bruker CryoProbe, located in the MIT

Chemistry Department Spectroscopy Laboratory.

Supporting Information Available: An appendix of equation

justifications and calculation derivations, synthetic details for

key intermediates, peptide NMR and MS characterization tables,

and AUC data. This material is available free of charge via the

Internet at http://pubs.acs.org.

JA0457462

(63) For a precedent applied to fast equilibration of ¹³C resonances, see: Park,

S.-H.; Shalongo, W.; Stellwagen, E. Proteins: Struct., Funct., Genet. 1998,

33, 167-178.

9

1704 J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005

Article Doi

Water-solubilized, cap-stabilized, helical polyalanines: Calibration standards for NMR and CD analyses

DOI: 10.1021/ja0457462

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Article abstract of DOI:10.1021/ja0457462

Full text of DOI:10.1021/ja0457462

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