Conformation-specific spectroscopy of capped, gas-phase Aib oligomers: Tests of the Aib residue as a 310-helix former

Article abstract of DOI:10.1039/c6cp04909e

The conformational preferences of a series of capped peptides containing the helicogenic amino acid aminoisobutyric acid (Aib) (Z-Aib-OH, Z-(Aib)₂-OMe, and Z-(Aib)₄-OMe) are studied in the gas phase under expansion-cooled conditions. Aib oligomers are known to form 3₁₀-helical secondary structures in solution and in the solid phase. However, in the gas phase, accumulation of a macrodipole as the helix grows could inhibit helix stabilization. Implementing single-conformation IR spectroscopy in the NH stretch region, Z-Aib-OH and Z-(Aib)₂-OMe are both observed to have minor conformations that exhibit dihedral angles consistent with the 3₁₀-helical portion of the Ramachandran map (φ, ψ = -57°, -30°), even though they lack sufficient backbone length to form 10-membered rings which are a hallmark of the developed 3₁₀-helix. For Z-(Aib)₄-OMe three conformers are observed in the gas phase. Single-conformation infrared spectroscopy in both the NH stretch (Amide A) and CO stretch (Amide I) regions identifies the main conformer as an incipient 3₁₀-helix, having two free NH groups and two C10 H-bonded NH groups, labeled an F-F-10-10 structure, with a calculated dipole moment of 13.7 D. A second minor conformer has an infrared spectrum characteristic of an F-F-10-7 structure in which the third and fourth Aib residues have φ, ψ = 75°, -74° and -52°, 143°, Ramachandran angles which fall outside of the typical range for 3₁₀-helices, and a dipole moment that shrinks to 5.4 D. These results show Aib to be a 3₁₀-helix former in the gas phase at the earliest stages of oligomer growth.

Full text of DOI:10.1039/c6cp04909e

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Conformation-specific spectroscopy of capped,
gas-phase Aib oligomers: tests of the Aib residue
as a 310-helix former†
Cite this:DOI: 10.1039/c6cp04909e
Joseph R. Gord,a Daniel M. Hewett,a Alicia O. Hernandez-Castillo,a
Karl N. Blodgett,a Matthew C. Rotondaro,b Adalgisa Varuolo,b Matthew A. Kubasik*b
and Timothy S. Zwier*a
The conformational preferences of a series of capped peptides containing the helicogenic amino acid
aminoisobutyric acid (Aib) (Z-Aib-OH, Z-(Aib)2-OMe, and Z-(Aib)4-OMe) are studied in the gas phase
under expansion-cooled conditions. Aib oligomers are known to form 310-helical secondary structures
in solution and in the solid phase. However, in the gas phase, accumulation of a macrodipole as the
helix grows could inhibit helix stabilization. Implementing single-conformation IR spectroscopy in the
NH stretch region, Z-Aib-OH and Z-(Aib)2-OMe are both observed to have minor conformations that exhibit
dihedral angles consistent with the 310-helical portion of the Ramachandran map (f, c = À571, À301),
even though they lack sufficient backbone length to form 10-membered rings which are a hallmark of the
developed 310-helix. For Z-(Aib)4-OMe three conformers are observed in the gas phase. Single-conformation
infrared spectroscopy in both the NH stretch (Amide A) and CQO stretch (Amide I) regions identifies the
main conformer as an incipient 310-helix, having two free NH groups and two C10 H-bonded NH groups,
labeled an F-F-10-10 structure, with a calculated dipole moment of 13.7 D. A second minor conformer has
an infrared spectrum characteristic of an F-F-10-7 structure in which the third and fourth Aib residues
have f, c = 751, À741 and À521, 1431, Ramachandran angles which fall outside of the typical range for
310-helices, and a dipole moment that shrinks to 5.4 D. These results show Aib to be a 310-helix former
in the gas phase at the earliest stages of oligomer growth.
Received 14th July 2016,
Accepted 25th August 2016
DOI: 10.1039/c6cp04909e
www.rsc.org/pccp
most commonly found secondary structure, it accounts for
only about 30% of residues in protein X-ray structures.8 Clearly,
I. Introduction
The intrinsic conformational propensities of amino acid residues the forces that favor the development of well-defined helical
for promoting (or disrupting) helical secondary structure have structures, such as intramolecular hydrogen bonding and inter-
been examined computationally1,2 and experimentally.3–6 The residue side-chain packing, are competing against other con-
great majority of proteinaceous helices determined in X-ray formational imperatives. In fact, the balance between these
crystal structures are right-handed a-helices,7,8 characterized by forces can sometimes be tipped by the presence of even a single
3.6 residues per turn and hydrogen-bonded rings of 13 atoms residue.11,12
(3.613-helices) closed by backbone hydrogen-bonding pairs
Aminoisobutyric acid (Aib) is a naturally occurring amino
between residues i + 4 - i and prototypical Ramachandran acid containing two side chain methyl groups. The symmetric
angles of f, c = À601, À451.7 About 10% of helices found in dimethylation of the a-carbon renders Aib achiral, similar
protein structures are more tightly-wound 310-helices, with 3.0 to glycine. In stark contrast to glycine, which is known to be
residues per turn and rings of 10 atoms closed by i + 3 - i the residue least supportive of helical structure,1,3–6,10,13 Aib
backbone hydrogen-bonding partners and Ramachandran residues are strongly helicogenic; oligomers of Aib are known
angles of f, c = À571, À301.8–10 Although the a-helix is the to induce formation of the 310-helix in solution and in the
condensed phase.13–16 The Aib residue is found singly and
contiguously (up to (Aib)4) in the naturally occurring peptaibol
family of antimicrobial peptides. The helicogenicity of con-
tiguous Aib residues is believed to allow peptaibols to form
helical transmembrane pores essential to their mechanism of
a Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
E-mail: Zwier@purdue.edu
b Department of Chemistry and Biochemistry, Fairfield University, Fairfield,
Connecticut 06824, USA. E-mail: MKubasik@fairfield.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp04909e antimicrobial activity.13,15,17
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The Amide A and Amide I vibrational modes of polar amide
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functional groups are particularly sensitive to the local electro-
static environment and are consequently diagnostic of protein
and peptide secondary structural features such as intramolecular
hydrogen bonding.18,19 Toniolo and coworkers monitored the
onset of helical secondary structure in a series of Aib homo-
peptides, Z-(Aib)n-OtBu (Z = benzyloxycarbonyl cap, n = 1–12), by
examining the NH stretch (Amide A) region of the infrared.14
Since the NH stretch fundamentals shift to lower frequency
with increasing strength of the hydrogen bonds in which they
are involved, they serve as an important diagnostic of structure,
especially in non-polar solvents where interference from solvent
is minimal.19 Solvated in deuterochloroform, Z-(Aib)n-OtBu exhi-
bits two amide A spectral bands, assigned to ‘‘free’’ and ‘‘intra-
molecularly hydrogen bound’’ NH stretches. With increasing ‘n’,
the ‘‘hydrogen bound’’ spectral band grows at the expense of
the ‘‘free’’ spectral band, converging to a limit of two free NH
stretches, characteristic of a 310-helical bonding pattern.
Fig. 1 The complete series of capped Aib homopeptides investigated in this
study. CQO groups have been numbered in red and NH groups have been
numbered in blue, in both cases from N- to C-terminus with 0 indicating the
CQO in the benzyloxycarbonyl cap (Z-cap).
calculated permanent dipole moments of only 2.6 and 2.8 D for
In a more recent study, Toniolo and Ge employed 2D IR the –OH and –NHMe structures, respectively,22 values smaller than
spectroscopy to investigate the CQO stretch (Amide I) spectro- the dipole moments of the individual peptide units (3.46 D). The
scopy of a series of Aib oligomers (Z-(Aib)n-OtBu, n = 3, 5, 8, 10) conformational flexibility of the glycine residue allowed the back-
dissolved in CDCl3.20 Although individual modes are not resolv- bone to adopt a configuration capable of supporting this mixed-
able in CDCl3, estimates of local Amide I frequencies and helical architecture, with C7 (i + 2 - i) H-bonds serving as spacers
coupling constants have been obtained through fits of the 2D between the remote C14 and C16 H-bonds.
IR spectral contours. The 2D IR spectra indicate that Amide I
Gas phase studies have also been carried out to evaluate the
spectral features characteristic of 310-helices emerge as early as competition among different hydrogen-bonding motifs in a
n = 5.20 The isotope-edited Amide I region linear infrared spectra series of tetrapeptides capable of forming a variety of intra-
of Kubasik and coworkers, employing 13C-enriched Z-(Aib)6-OtBu molecular hydrogen bonds.23 Mons and coauthors saw compe-
isotopologues, reveal localized 13CQO Amide I modes as distinct tition among structures involving C10 and C7 hydrogen-bonded
spectral features at reduced frequencies—an improvement over rings, NHÁÁÁp hydrogen-bonded interactions, and 27-ribbon struc-
the broad, unresolved 12CQO Amide I band.21 These isotope- tures in their study of Ac–Phe–Ala–Ala–NH2, Ac–Ala–Ala–Phe–NH2,
edited spectra confirm a 310-helical hydrogen-bonding pattern and Ac–Ala–Phe–Ala–NH2.
for Z-(Aib)6-OtBu, since the lowest frequency 13C-localized Amide I
Additionally, Mons and co-workers have examined a tetra-
oscillators are those that, within a 310 helical hydrogen-bonding peptide containing a phenylalanine residue flanked by two Aib
pattern, are both hydrogen bond acceptors (at the carbonyl oxygen) residues (Ac–Aib–Phe–Aib–NH2) under jet-cooled conditions.24
and hydrogen bond donors (at the amide group hydrogen). Its main conformer exhibited NH stretch transitions due to a
Although 13C-enrichment shifts a 13C-Amide I band to lower pair of C10 H-bonds that are indicative of an incipient 310-helix,
frequency, the spectral features in the Amide I and Amide A appearing at 3374 cmÀ1 and 3405 cmÀ1 22,24,25
regions are still inherently broadened by solvent effects. The goal of the present study is to investigate more fully the
.
Under jet-cooled conditions, gas phase spectroscopic studies folding propensities and spectroscopic signatures of the series of
of peptides offer superior spectral resolution as compared to capped Aib homopeptides shown in Fig. 1, under cold, isolated
solvent-broadened condensed phase efforts. Additionally, gas conditions in the gas phase. These structures will be referred to in
phase studies examine the intrinsic conformational propensities short-hand as Z-(Aib)n-X (n = 1, 2, 4 and X = OH [for n = 1 only] or
of amino acid residues and peptides by removing subtle structure- OMe). The benzyloxycarbonyl (Z) cap provides the necessary UV
supporting aspects of a solvent environment. Furthermore, in the chromophore for carrying out single-conformation spectroscopy,
gas phase, the NH stretch region can be used as a structural while retaining a pure Aib homopeptide backbone. The present
diagnostic, reporting directly on the hydrogen-bonding architec- work examines the intrinsic conformational propensities of the
ture of the conformations, with no interference from solvent Aib residue and contiguous Aib units, elaborated as short homo-
absorption (e.g., due to H2O).
oligomers. By cooling the molecule in a supersonic expansion, the
In a recent study of the glycine homopeptide series Z-(Gly)n-OH present work probes the lowest energy conformations of these
with n = 1, 3, 5 and Z-(Gly)5-NHMe (where Z is a benzocarboxy cap, homopeptides in their isolated form, unperturbed by solvent or
Fig. 1), Dean et al. showed that both of the n = 5 oligomers folded solid state intermolecular interactions. Single-conformation IR
exclusively into 14/16 mixed helices in which the hydrogen bonds spectra are compared to the predictions of DFT calculations,
associated with 14-membered H-bonded rings (labeled C14) leading to assignments that identify the preferred conformations
and 16-membered rings (C16) point in opposite directions of the molecules in this series, and spectroscopically characterize
along the peptide backbone. The assigned structures possess them in the ultraviolet (UV) and infrared (IR).
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II. Methods
A. Experimental methods
for the UV laser to ionize is decreased, and a dip in the total ion
signal is observed. By performing active baseline subtraction
on each pair of ‘‘IR on’’ and ‘‘IR off’’ peaks with a Stanford
Research Systems gated integrator (SR-250), a spectrum can be
recorded in which the fractional depletion in ion signal is
plotted as a function of IR frequency.
Once the R2PI and RIDIR spectra have been recorded, IR-UV
hole-burning (HB) spectroscopy can be used to separate the
R2PI spectrum into a set of conformer-specific UV spectra.32 To
collect these IR-UV HB spectra, the wavelength of the IR laser is
fixed on a vibrational transition belonging to a specific con-
formation, and the wavelength of the UV laser is scanned. By
implementing the same timing and overlap schemes described
in RIDIRS, and performing active baseline subtraction, the
fractional depletion of the ion signal versus UV frequency can
be recorded to obtain single-conformation UV spectra.
Samples were laser desorbed into the gas phase from the surface
of a graphite rod using the 1064 nm output from a Nd:YAG laser
(Continuum MiniLite).26 Laser pulses were approximately 6 ns
in duration with 3–5 mJ of energy per pulse. The desorption
rod was translated linearly using a pusher motor during data
collection to ensure that fresh sample was available throughout
each scan. This laser desorption scheme has been described
in detail elsewhere.22,27 After being desorbed, the gas-phase
sample molecules were entrained in an expansion of argon gas
coming from a Parker Fluidics, Series 9 general valve with a
500–800 mm nozzle diameter operating at 20 Hz with approxi-
mately 5.5 bar of backing pressure. Sample molecules were
then collisionally cooled in a supersonic expansion, funneling
their population primarily into the zero-point vibrational energy
levels of the most stable conformers.28–30 Downstream, the seeded
expansion was passed through a 3 mm skimmer to form a
molecular beam which was interrogated using single- and
double-resonance laser schemes in the ionization region of a
Wiley McLaren, time-of-flight mass spectrometer.31
Resonant two-photon ionization (R2PI) was used to record
the electronic excitation spectra for the molecules of interest.
Scans over the 35 000 cmÀ1 to 38 000 cmÀ1 region were recorded
using the doubled output of a Radiant Dyes, NarrowScan dye
laser pumped by 355 nm light from a Continuum, SureLite II,
Nd:YAG laser. When UV light from the dye laser is resonant
with a vibronic transition between the S0 and S1 states of a
given conformation, a first photon is absorbed which promotes
the molecule to the excited state; upon immediate absorption
of a second photon, the molecule is ionized. By tuning the UV
laser over a range of wavelengths characteristic of the Z-cap UV
chromophore, the vibronic spectrum of all conformations pre-
sent in the jet can be recorded.
A KTP/KTA-based infrared parametric converter (LaserVision)
was used to generate IR light in the NH and OH stretch regions
(3200 cmÀ1 to 3700 cmÀ1) with typical output energies on the
order of 12–15 mJ per pulse. When desired, a AgGaSe2 crystal was
used to difference frequency mix into the mid-infrared, in order
to generate light in the 1600 cmÀ1 to 1800 cmÀ1 region which
encompasses the Amide I (CQO stretch) fundamentals.32 Typical
output energies in the Amide I region were on the order of
500–1000 mJ per pulse.
Single-conformation IR spectra were recorded using reso-
nant ion-dip IR spectroscopy (RIDIRS).32,33 In RIDIRS, the UV
laser, operating at 20 Hz, has its wavelength fixed on a vibronic
transition belonging to a single conformer, selected from the
R2PI spectrum, in order to generate a constant level of ion
signal. The 10 Hz, infrared beam is spatially overlapped with,
but temporally precedes the UV beam. When the IR laser is
resonant with a vibrational transition belonging to the same
conformation that is being pumped with the UV laser, some of
the population is moved out of the vibrational zero-point level
B. Computational methods
The results of Density Functional Theory (DFT) calculations are
used to assist in making structural assignments of the observed
IR spectra. To obtain these computational results, a starting
structure is submitted to a conformational search performed at
the Amber* force field34 level within a 100 kJ molÀ1 energy
window using MacroModel (Schrodinger, Inc.).34,35 Structures from
¨
the conformational search with energies within B50 kJ molÀ1
of the force field global minimum are re-optimized to calculate
geometries and harmonic frequencies and IR intensities via Density
Functional Theory (DFT) calculations (Gaussian0936) using the
M05-2X functional and a 6-31+G(d) basis set.37 The vibrational
frequencies are scaled using well established scale factors of
0.940 in the NH and OH stretching regions and 0.960 in the
Amide I region for this level of theory.22,38–40 We would like to
point out that there is also an opportunity to apply a slight
correction (B10 cmÀ1) to the amide NH stretch frequencies to
account for stretch-bend Fermi resonance; however we have not
done so here.41 Relative energies of the conformers, including
corrections for zero-point energy effects at the harmonic level,
are included in the figures and tables as appropriate.
C. Synthetic methods
Commercially available starting materials Aib (Acros), Z-Aib-OH
(Bachem) and HClÁAib-OtBu (Bachem) were used as received.
HClÁAib-OMe was prepared from MeOH and trimethylsilyl
chloride.42 Aib dimers, Z-(Aib)2-OtBu and Z-(Aib)2-OMe, were
prepared using EDCI as a coupling reagent at 60 1C in a CEM
Discover SP microwave reactor for 30 minutes.43 These two
dimers were used to prepare Z-(Aib)4-OMe via literature methods.14
Z-(Aib)2-OMe and Z-(Aib)4-OMe were purified via flash silica gel
chromatography.
D. Nomenclature
Analysis of the single-conformation data employs the amide
group numbering scheme shown in Fig. 1. In this scheme, NH
groups are labeled 1–4 and CQO groups are numbered from
and promoted to a vibrationally excited level within the ground 0–4 beginning at the N-terminal carbamate group of the Z-cap
electronic state, thus the total number of molecules available and extending to the C-terminus. Hydrogen-bonded rings are
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named according to the number of atoms in the cycle closed by IR assignments discussed below. The origin of conformer A is
the H-bonding interaction. For example, a ten-atom cycle formed shifted just 1 cmÀ1 from the origin transition for Z-Gly-OH
by an intramolecular hydrogen bond is labeled as ‘C10’. To studied by Dean et al. (37 601 cmÀ1). Additionally, the assigned
describe the hydrogen-bonding architecture of the conformations, vibronic activity is nearly identical to that for Z-Gly-OH suggesting
a notation analogous to that used by Mons and co-workers44 is that the two structures are very similar, with nearly equivalent
implemented in which the hydrogen-bonding behavior of NH environments for the Z-cap.22
groups will be represented from N- to C-terminus by either ‘‘F’’
Resonant ion-dip infrared spectroscopy was used to record
meaning a free NH, uninvolved in an intramolecular hydrogen conformer-specific IR spectra in the NH and OH stretch regions
bond, or ‘‘n’’, where ‘‘n’’ is the number of atoms in the resulting for both conformers of Z-Aib-OH. Assignments are made by com-
hydrogen-bonded ring. So, for example, a structure having 4 NH paring experimental spectra with calculated, scaled, harmonic
groups might be described as F-F-10-10 meaning the NH groups frequencies and IR intensities, depicted as stick spectra, in
from N- to C-terminus are free, free, C10, and C10. It should be Fig. 2(b). The RIDIR spectrum for conformer A shows two well-
noted that, due to the achiral nature of the Aib residue, the resolved transitions in the NH/OH stretching region. Comparison
structures shown in the figures and referred to in the tables have to computational results allows us to assign the transition at
mirror images that are identical in energy and spectral features. 3447 cmÀ1 to a C5 hydrogen-bonded NH group. The second
The mirror image structures have backbone angles of opposite transition, located at 3579 cmÀ1, is assigned to a free OH stretch
sign to those reported in the table(s).
in the carboxylic acid group. The RIDIR spectrum for the minor
conformer B is unusual in that it has two broadened transitions
centered near those of conformer A as well as a pair of sharp
transitions at 3468 cmÀ1 and 3583 cmÀ1 (marked with daggers
in Fig. 2(b)). It is likely that the frequency of the UV monitor
transition has contributions both from conformer B (responsible
III. Results
A. Z-Aib-OH
Resonant two-photon ionization (R2PI) and IR-UV hole-burning for the sharp IR transitions) and from a hot band of conformer A
spectra for Z-Aib-OH are shown in Fig. 2(a). The R2PI spectrum (responsible for the broadened bands centered on the transitions
contains a single dominant feature at 37 602 cmÀ1 and two due to conformer A). The small shift and broadening in fre-
minor features at 37 576 cmÀ1 and 37 619 cmÀ1. The IR-UV HB quency is consistent with excitation out of a vibrationally excited
spectra identify the 37 602 cmÀ1 and 37 576 cmÀ1 transitions as level of a low-frequency vibration, which can shift and modulate
S0–S1 origin transitions for two distinct conformations of Z-Aib-OH, the NH stretch frequency. The small depletions due to B are also
labeled A and B, respectively. The peak at 37 619 cmÀ1 is identified consistent with this overlap in the UV, where a portion of the ion
as a vibronic band, tentatively assigned to a butterfly motion of signal is due to ‘A’, and therefore not available for depletion on
conformer A, involving the ring and backbone, based upon our resonances due to ‘B’.
Fig. 2 (a) R2PI (black) and IR-UV hole-burning spectra (red for A and blue for B) for Z-Aib-OH. Hole-burning spectra were recorded with the IR hole-burn
laser fixed at 3579 cmÀ1 for A, and 3468 cmÀ1 for B. (b) RIDIR spectra for conformers A and B of Z-Aib-OH in the NH and OH stretch region. Scaled,
harmonic, vibrational frequencies and infrared intensities for the assigned structures are shown as stick spectra below the experimental data, calculated
at the DFT M05-2X/6-31+G(d) level of theory. The daggers indicate transitions specific to conformer B. Asterisks indicate frequencies used for IR-UV
hole-burning experiments. See text for further discussion.
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The assigned structures for conformers A and B are shown carboxylic acid group and any other hydrogen-bonding sites
in Fig. 3, and key structural parameters are summarized in along the backbone, we surmise that the methyl ester derivative
Table 1. Conformer A is calculated to be the global minimum, (not studied here) would likely adopt similar structures, enabling
among all computed structures, and contains an extended C5 direct comparison of the present Z-Aib-OH results with the
interaction between the NH and CQO groups that span the methoxy-capped Z-(Aib)n-OMe molecules with n = 2, 4.
Aib residue with f, c = 1801, 1801 and a cis carboxylic acid
B. Z-(Aib)2-OMe
OH group. Conformer B is assigned to a significantly different
structure in which neither of the CQO groups nor the NH The R2PI and IR-UV HB spectra for Z-(Aib)2-OMe are shown in
group engage in hydrogen bonding. Instead, the steric crowding Fig. 4(a). Most of the intensity in the R2PI spectrum appears
of the dimethylated a-carbon causes the two amide groups to below 37 500 cmÀ1, significantly lower in energy than either of
orient with f, c = À491, À371, values close to those found in a the origins for the two conformers of Z-Aib-OH (37 602 and
310-helix.10 Additionally, parallel alignment of the CQO groups 37 576 cmÀ1). IR-UV hole-burning spectra identify three con-
produces a dipole moment that is twice the magnitude observed formations labeled A, B, and C shown in red, blue, and green,
for conformer A (3.3 D in conformer B vs. 1.5 D in conformer A). respectively. The S0–S1 origin of conformer A is assigned to the
Since neither conformer contains an interaction between the peak at 37 436 cmÀ1. The hole-burning spectra for conformers B
and C were a challenge to record due to the small intensity of
their transitions relative to those of conformer A, requiring
significant signal averaging to achieve an acceptable signal-to-
noise ratio. Therefore, it is difficult to identify the S0–S1 origin of
conformer B with certainty, and the transition at 37545 cmÀ1
,
the first transition relatively free of interference that shows up
clearly in the R2PI spectrum, was used for RIDIRS. The S0–S1
origin of conformer C at 37604 cmÀ1, is within 2 cmÀ1 of the
origin for conformer A of Z-Aib-OH, indicating that the phenyl
ring is likely located in an environment similar to the phenyl
ring environment in conformer A of Z-Aib-OH.
The R2PI spectrum for Z-(Aib)2-OMe is much more congested
than that of Z-Aib-OH, due to the presence of several conformers
and/or much stronger Franck–Condon activity in low-frequency
modes. Specifically, a clear progression of peaks is seen in conformer
A, beginning at 37 436 cmÀ1, with members separated by approxi-
mately 13 cmÀ1. Vibrational assignments, discussed below, indicate
that this progression is caused by an in-phase rocking motion
involving the phenyl ring and the C-terminal end of the backbone.
Fig. 4(b) presents RIDIR spectra of the three conformers of
Z-(Aib)2-OMe, recorded over the 3300–3500 cmÀ1 region where
the amide NH stretch fundamentals are known to appear.
Methoxy substitution removes the carboxylic acid OH, so scans
above 3500 cmÀ1 were not necessary. The three IR spectra
show distinct patterns characteristic of three structural types.
Calculated (M05-2X/6-31+G(d)), scaled (0.940), harmonic fre-
quencies and IR intensities are shown below the experimental
data as stick spectra.
Fig. 3 (a) Assigned structure for conformer A of Z-Aib-OH. The dotted red
line indicates a C5 hydrogen-bonding interaction. (b) Assigned structure for
conformer B of Z-Aib-OH. Structural families, computed (M05-2X/6-31+G(d))
relative energies (kJ molÀ1), and dipole moments (D) for the two conformers
are listed.
Table 1 Dihedral angles for all assigned conformations.a All angles are given in degrees. Relative energies are given in kJ molÀ1
Molecule
Relative energy (kJ molÀ1
)
H-bonding pattern Ring orientation f (1) c (1) f (2) c (2) f (3) c(3) f (4) c (4)
Z-Aib-OH (A)
Z-Aib-OH (B)
0
4.5
0
7.2
5.2
0.0
3.5
3.9
24.4
5
F-F
F-5
F-7/5
83
84
71
77
84
74
74
69
92
À180 À180
49
À57
À72
37
Z-(Aib)2-OMe (A)
Z-(Aib)2-OMe (B)
Z-(Aib)2-OMe (C)
Z-(Aib)4-OMe (S)
Z-(Aib)4-OMe (NS)
Z-(Aib)4-OMe (B)
Z-(Aib)4-OMe (C)
À38 À178 À179
70
180
180
À55
À57
À58
68
179
180
F-F
180 À180
F-F-10-10
F-F-10-10
F-F-10-7
14-7-7-F
À61
À61
À63
À82
À26
À27
À25
34
À27 À56 À31
51
40
À22 À57 À35 À48 À40
À30
75 À74 À52
151 43
143
49
À79 À53
a
Due to the achiral nature of Aib, a second set of equivalent conformations exists in which the signs of all the dihedral angles are changed.
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Fig. 4 (a) R2PI (black) and IR-UV hole-burning spectra for conformers A, B, and C of Z-(Aib)2-OMe. The hole-burning spectra were recorded with the IR
hole-burning laser fixed at 3418 cmÀ1 for A, 3342 cmÀ1 for B, and 3407 cmÀ1 for C, marked with asterisks in (b). (b) RIDIR spectra for conformers A, B, and
C of Z-(Aib)2-OMe in the NH stretch region. Scaled, harmonic, vibrational frequencies and infrared intensities for the assigned structures calculated at the
M05-2X/6-31+G(d) level of theory are shown as stick spectra below experiment. Transitions used to record RIDIR spectra are marked with asterisks in (a).
The RIDIR spectrum of conformer A shows two well-resolved transitions of A, indicating a small overlap with a vibronic
NH stretch fundamentals at 3418 cmÀ1 and 3472 cmÀ1. The transition of conformer A at the UV wavelength chosen. Unique IR
higher-frequency transition at 3472 cmÀ1 is identified as the transitions at 3343 cmÀ1 and 3461 cmÀ1 are due to conformer B.
free stretch of the NH of the carbamate group resulting from The higher-frequency transition at 3461 cmÀ1 is assigned to the
the Z-cap. The lower-frequency peak at 3418 cmÀ1 is assigned to free (N-terminal) NH group, while the lower-frequency 3343 cmÀ1
a C5 hydrogen-bonded NH stretch fundamental of the interior peak is assigned to the central amide NH group that engages
amide group. This peak is shifted significantly (À29 cmÀ1) from simultaneously in C7 and C5 hydrogen bonds with the Z-cap
the position of the assigned C5 NH stretch in the IR spectrum (carbamate) carbonyl and the C-terminal methyl ester carbonyl,
of Z-Aib-OH conformer A. The C5 structure of Z-Aib-OH con- respectively. The frequency of this NH stretch is close to that of
tains an NH from a carbamate group, while the C5 structure of other pure C7 hydrogen-bonded rings.22,45 The stick diagram
Z-(Aib)2-OMe, conformer A, contains an NH of an amide group. shows the calculated, scaled spectrum that best matches the
Conformer A is thus assigned to the F-5 structure shown in experimental spectrum, arising from an F-7/5, bifurcated double-
Fig. 5(a).
In the IR spectrum of conformer B, small transitions marked
ring structure, shown in Fig. 5(b).
The IR spectrum for conformer C contains two clearly resolved
with daggers in Fig. 4(b) appear at frequencies due to the two NH stretch fundamentals appearing at 3407 cmÀ1 and 3427 cmÀ1
.
Fig. 5 (a), (b), and (c) show the assigned structures for conformers A, B, and C of Z-(Aib)2-OMe. Hydrogen bonds are indicated with dotted red lines.
Structural families, relative energies (kJ molÀ1), and dipole moments (D) are listed.
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The computed structure that offers the best match to this
experimental spectrum exhibits two C5 hydrogen-bonded NH
groups. As previously observed, the phenyl ring adopts an
orientation similar to the extended structure (conformer A) of
Z-Aib-OH, consistent with the electronic origins of these two
structures being nearly identical. This structure is assigned as a
fully-extended 5-5 structure. The N-terminal (carbamate) NH
group is assigned to the lower-frequency transition at 3407 cmÀ1
,
and the central amide NH group is assigned to the higher-
frequency transition at 3427 cmÀ1. This fully extended structure
minimizes interaction between the side chains, backbone, and
phenyl ring and has a dipole moment of 4.6 D. This is in line
with the dipole moments calculated by Dean et al. for analogous
extended structures of Z-Glyn-OH.22
The assigned structures for conformers A–C, shown in Fig. 5,
possess the f and c dihedral angles summarized in Table 1.
It is noteworthy that the F-5 structure of conformer A, which is
the global minimum structure, possesses Ramachandran angles
(f, c = À571, À381) about Aib[1] that are near those found in an
idealized 310-helix (f, c = À571, À301). The Ramachandran
angles of Aib[2] allow for a C5 hydrogen-bonded structure. This
same C-terminal C5 structure is found in the other two con-
formers, most likely because the C-terminal methyl ester
possesses no NH group to participate in hydrogen bonding.
The only other unique Ramachandran angles are those asso-
ciated with the C7 g-turn (f1, c1 = +721, À701), which despite its
strong H-bond (nNH = 3342 cmÀ1) has an energy +7.2 kJ molÀ1
above the F-5 structure.
Fig. 6 R2PI (black) and IR-UV hole-burning spectra for conformers A (red)
and B (blue) of Z-(Aib)4-OMe. The hole-burning spectra were recorded
with the IR hole-burning laser fixed at 3406 cmÀ1 for A, and 3383 cmÀ1 for B,
marked with asterisks in Fig. 7(b).
its transitions. Instead, conformer C was identified by its
The significantly shifted electronic origins of conformers possession of a unique IR spectrum.
A and B as well as the extensive low-frequency vibronic activity
RIDIR spectra of all three conformations collected in the
observed in their R2PI spectra lend additional support to CQO and NH stretching regions are shown in Fig. 7(a) and (b),
the conformational assignments in Fig. 5. Both structures fold respectively. As with the peptides examined above, calculated
the C-terminal end of the peptide back over the Z-cap, enabling (M05-2X/6-31+G(d)), scaled (0.940), harmonic frequencies and
a strong interaction so that upon p–p* excitation, Franck– infrared intensities for the assigned conformers are shown below
Condon activity is evident, likely involving the low-frequency each experimental spectrum. A good spectral match between a
motions of the peptide backbone, (n B 13 cmÀ1) against the computed spectrum for a low-energy conformer and the experi-
aromatic ring.
mental spectrum of conformer A of Z-(Aib)4-OMe with four NH
stretch transitions, occurring as two closely-spaced pairs, allows
us to assign two lower-frequency H-bonded NH fundamentals
C. Z-(Aib)4-OMe
Fig. 6 presents the R2PI and IR-UV hole-burning spectra of (3383 cmÀ1 and 3406 cmÀ1) and two free NH stretch funda-
Z-(Aib)4-OMe. The R2PI spectrum for Z-(Aib)4-OMe shows signi- mentals (3464 cmÀ1 and 3478 cmÀ1).
ficantly less congestion than that of Z-(Aib)2-OMe. Even so,
The calculated spectrum for the assigned structure of conformer
three unique conformations having origin transitions located A associates a pair of C10-hydrogen-bonded NH groups with the
at 37 581 cmÀ1, 37 434 cmÀ1, and 37 629 cmÀ1 were identified low-frequency pair (3383 cmÀ1 and 3406 cmÀ1), classifying the
based on hole-burning data, and are labeled A, B, and C, structure as F-F-10-10. The H-bonded NH stretch fundamentals
respectively. Based on the intensities of the S0–S1 origin transi- appear within a few cmÀ1 of those found for the C10/C10 pair in
tions in the R2PI spectrum, conformer A appears to be the the F-F-10-10 structure of Ac–Aib–Phe–Aib–NH2 studied by Mons
dominant conformation. The origin transition for conformer B and co-workers.24 This pattern of transitions indicates the first
appears only 2 cmÀ1 lower than the A origin in Z-(Aib)2-OMe, stages of a prototypical 310-helical structure in Z-(Aib)4-OMe. Indeed,
suggesting a similar phenyl ring environment. Single-conformer now all four Aib residues adopt local configurations characteristic
UV spectra (lower traces of Fig. 6) were obtained for conformers of a full turn of a 310-helix (Table 1), with Ramachandran angles
A and B. (Note that the feature in the IR-UV HB spectrum of (f, c) = (À611, À271), (À571, À221), (À571, À351), and (À481, À401).
conformer B which appears at a frequency identical to the The slight variation of the f, c angles of the C-terminal Aib residue
origin transition of conformer A is due to incomplete subtrac- is not surprising in light of the fact that the C-terminal ester group,
tion in the gated integrator.) However, a similar spectrum for lacking the capacity to function as a hydrogen-bonding donor, does
conformer C was not available due to the weak intensity of not support the formation of additional hydrogen bonds.
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Fig. 7 RIDIR spectra for conformers A, B, and C of Z-(Aib)4-OMe in the (a) Amide I region and (b) NH stretch region. Asterisks indicate the transitions
used for IR-UV hole-burning in Fig. 6. Calculated, scaled, harmonic, vibrational frequencies and infrared intensities for the assigned structures are shown
as stick spectra below experiment.
Our calculated assignment for conformer A of Z-(Aib)4-OMe frequency than a free, interior CQO, even though it participates
is supported by the IR spectrum in the Amide I region shown in in a hydrogen bond.
Fig. 7(a), which shows two closely spaced sets of transitions
occurring at 1701 cmÀ1/1707 cmÀ1 and 1727 cmÀ1/1739 cmÀ1
The range of calculated frequencies and intensities for the
Amide I normal modes shown in Fig. 7(a) reflects the variety of
,
as well as a single higher-frequency stretch at 1762 cmÀ1. We local electrostatic environments surrounding individual amide
note that these Amide I stretches (as well as those for confor- oscillators as well as the inter-amide coupling between local
mers B and C of Z-(Aib)4-OMe, discussed below) are shifted to oscillators that gives rise to delocalized Amide I normal modes.
higher frequency than similar oscillators observed for proteins Our gas phase spectra and their conformational assignments
in the solution phase.46,47 This is consistent with measure- provide good tests for Hessian analysis, which seeks to extract
ments made in the gas phase and is in accord with what has local mode frequencies and inter-amide coupling constants.48
been reported previously for similar systems.22,48 Assignments Additionally, our solvent-free system could provide verification
in the Amide I region are complicated by the different func- and/or calibration of the growing number of electrostatic maps,
tional groups (carbamate, amide, and ester) containing a CQO, which have been developed for the purpose of Amide I band
and the coupling between CQO groups in the helix, which spectral prediction.49
delocalizes the vibrational modes across more than one carbonyl.
The RIDIR spectrum of conformer B in the NH stretch
The labels in the figure identify the dominant contributor to region is shown as the middle trace (blue) in Fig. 7(b). A good
each transition and the resulting hydrogen-bonded structure. match with the calculated NH frequencies of an energy-
These assignments match nicely with our established under- minimized structure allows us to assign two higher-frequency,
standing of the Amide I region. Specifically, CQO groups free NH stretch transitions, 3454 cmÀ1 and 3463 cmÀ1, a single
involved in hydrogen bonding are shifted down in wavenumber C10 H-bonded NH stretch transition, 3380 cmÀ1, and a C7
from their otherwise free position. Note that ester and carbamate hydrogen-bonded NH to the strongly shifted and broadened set
groups have CQO stretch fundamentals that are inherently higher of peaks near 3250 cmÀ1. The calculated stick spectrum and the
in frequency by B50 cmÀ1 than their amide CQO counterparts.20 experimental spectrum match well, leading to an assignment of
This difference in site frequencies of the various functional conformer B to the F-F-10-7 structure shown in Fig. 8(b). The
groups explains why a carbamate CQO appears at a higher higher-frequency free NH stretch, 3464 cmÀ1, is due to the
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N-terminal carbamate NH. The transition at 3250 cmÀ1 is assigned a comparatively high frequency feature, despite its participation
to an NH participating in a C7 H-bond, which is calculated to have in hydrogen bonding.
an NHÁ Á ÁOQC hydrogen-bond distance of just 1.94 Å, leading
to its large shift of more than 120 cmÀ1 below the C10 NH in the NH stretch region is shown in green in Fig. 7(b). The
stretch. This structure is intriguing, as it shares much in spectrum is unique, with a single free NH stretch at 3462 cmÀ1
Finally, the RIDIR spectrum for conformer C of Z-(Aib)4-OMe
,
common with the F-F-10-10 structure of conformer A, but two NH groups engaged in intermediate-strength H-bonds,
formally exchanges a final C10 H-bond for a C7. We will return at 3355 cmÀ1 and 3382 cmÀ1, and an NH group in a strong
in the discussion to a further consideration of this structure H-bond at 3270 cmÀ1. The structure tentatively assigned to
and its relationship to the 310-helix.
conformer C is shown in Fig. 8(c), labeled as 14/11-7-7-F, with a
The good match between the experimental IR spectrum of predicted IR spectrum given in the stick diagram below the
conformer B in the Amide I region, Fig. 7(a), with the calculated experimental spectrum. The transition occurring at 3270 cmÀ1
spectrum of the energy minimized structure confirms the is due to the strong C7[NH1] H-bond. The pair of transitions at
assignment of an F-F-10-7 structure. The lowest frequency 3355 cmÀ1 and 3382 cmÀ1 are delocalized combinations of the
CQO stretch transition at 1681 cmÀ1 belongs primarily to the C14/C11 bonded NH and the NH involved in the weaker of the
strongest hydrogen-bonded CQO[2] which is involved in a tight two C7 rings. The Amide I region of conformer C, Fig. 7(a), is
C7 hydrogen-bonded ring. This oscillator is also coupled to less well-resolved than Amide I regions of the other two con-
the free CQO stretch of the adjacent CQO[3] at 1710 cmÀ1. The formers, having four broad bands centered around 1690 cmÀ1
,
band at 1729 cmÀ1 is a free, interior CQO[1]. The C10 carbamate 1712 cmÀ1, 1728 cmÀ1, and 1752 cmÀ1. The missing fifth band
CQO[0] and free, ester CQO[4] are the two highest frequency is likely hidden under the breadth of one of these other, much
Amide I fundamentals appearing at 1754 cmÀ1 and 1770 cmÀ1
.
broader bands. The observed transitions are consistent with the
As discussed previously, the carbonyl stretch of the carbamate is stick diagram predicted for the assigned structure, which predicts
Fig. 8 Assigned structures for conformers A, B, and C of Z-(Aib)4-OMe. Both the Schellman and non-Schellman versions of conformer A have been
shown. The dotted circle indicates the location of the Schellman helix reversal. Hydrogen-bonding interactions are shown with dotted red lines.
Structural families, relative energies (M05-2X/6-31+G(d), kJ molÀ1), and dipole moments (D) are listed. Non-amide hydrogen atoms have been removed
for clarity.
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the major CQO groups responsible for each absorption shown
in the figure.
IV. Analysis and discussion
The goal of this study is to evaluate the structural preferences of
a series of Aib-based homopeptides of increasing size in the gas
phase to determine whether their 310-helix forming propensity
carries over from solution to the gas phase. We seek to extract
and evaluate the spectroscopic signatures of the 310-helix, if it is
formed, identify possible competing structures, and understand
how the Aib residue influences them.
Our Aib homopeptides (Z-Aib-OH, Z-(Aib)2-OMe, and Z-(Aib)4-
OMe) vary in their propensity to adopt dihedral angles in the
310-helical region of the Ramachandran plot (f B À571,
c B À301), and in their capacities to form a C10 hydrogen
bond (i.e., i - i + 3 hydrogen bond of a Type III b-turn) that is
the structural hallmark of the 310-helix. For example, while
the Z-Aib-OH molecule is incapable of forming a C10 intra-
molecular hydrogen bond, a minor conformer of Z-Aib-OH is
seen to adopt Ramachandran angles that support such hydro-
gen bonds in larger members of the series. Conformer A of
Z-(Aib)2-OMe also displays a set of dihedral angles for the first
Aib residue that are near those of a 310-helix, (f, c = À571, À301),
even though it also lacks the backbone length necessary to make
a fully formed turn of the 310-helix. Indeed, the (f, c) angles of
the C-terminal Aib of conformer A of Z-(Aib)2-OMe are fully
extended (f B À1801, c B À1801). Finally, the Z-(Aib)4-OMe
molecule, large enough to allow two intramolecular C10 hydro-
gen bonds, adopts a clear 310-helix as its dominant conformer, in
which two C10 hydrogen bonds are formed, and all four residues
adopt dihedral angles characteristic of the 310-helix (Table 1).
Competing structures that incorporate C7, C11, and even C14
hydrogen-bonded rings form architectures classified as stalled or
mixed helices, which make minor contributions to the R2PI
spectrum. Even so, it is clear that in the gas-phase, free from
intermolecular stabilization and solvent effects, Aib is a robust
310-helix former.
Fig. 9 Energy level diagram for the relative, zero-point corrected energies
(kJ molÀ1) of the conformational minima of Z-Aib-OH (left) and Z-(Aib)2-OMe
(right) calculated at the M05-2X/6-31+G(d) level of theory. The conforma-
tional families of Z-Aib-OH are bracketed and labeled. The conformational
families of Z-(Aib)2-OMe are labeled below their respective columns.
4.5 kJ molÀ1 higher in energy. DFT calculations at the
M05-2X/6-31+G(d) level of theory show two intervening confor-
mers, plotted in the diagram, that are not observed experimentally.
If the calculated energy ordering is correct, these two structures
must have low energy barriers to isomerization to A, allowing
their population to be removed during the cooling process in
the expansion.
Not surprisingly, the energy level diagram for Z-(Aib)2-OMe,
displayed on the right-hand side of Fig. 9, shows a larger number
of low-energy structures than the diagram for Z-Aib-OH. Struc-
tural families are grouped by H-bonded features into columns;
individual structures are ordered according to relative energy.
A significant number of the calculated structures contain one
free NH group and one hydrogen-bonded NH group. The
observed H-bond types include: (i) a C5 ring formed between
the NH and CQO groups on a given Aib residue, characteristic
A. Energetics analysis
An energy level diagram comparing the calculated structures of an extended peptide backbone, (ii) a C7 ring in which an NH
for Z-Aib-OH is shown in the left-most column of Fig. 9. The group makes a more traditional, end-on interaction with the
structures assigned for conformers A and B are labeled. The CQO of the adjacent Aib residue, and (iii) in a few cases, a
limited backbone length of Z-Aib-OH prohibits the formation of special C5 in which the NH group of the second Aib residue
more complex hydrogen-bonding interactions, and all but four interacts with the lone-pair on the backside of the backbone,
of the calculated structures fall within a 13 kJ molÀ1 energy methoxy-group oxygen. There is a single structure, assigned to
window, containing only free OH groups and free or C5 hydrogen- conformer C, which contains two C5 hydrogen-bonded NH
bonded NH groups. The four structures falling outside this groups in a fully-extended peptide backbone geometry. In this
window are all at least 35 kJ molÀ1 above the calculated sense, conformer C is the sole member of another conforma-
minimum energy structure and possess hydrogen bonding tional family (5-5), and could rightfully have its own column in
interactions between either the NH group or the OH and the the diagram, but has been grouped with the F-5 structures for
p-cloud on the phenyl ring. These geometries induce substantial efficient space utilization.
strain on the backbone. Conformer A, with a single C5 intra-
Conformer A, an F-5 structure, is found to be the global
molecular hydrogen bond, was found to be the global minimum. minimum, with its first Aib residue having Ramachandran angles
Conformer B, with no intramolecular hydrogen bonding, is only close to that of a 310 helix (Table 1). It is noteworthy that
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this structure, incorporating a single C5 ring, is lower in energy columns have well-defined intramolecular hydrogen-bonding
by 5.2 kJ molÀ1 than conformer C, the fully extended structure, patterns; however the three columns to the right simply group the
with its two weak C5 H-bonds. This energy stabilization of A structures by the number of amide-amide H-bonds they possess.
over C must be attributed, at least in part, to the preference
of the first Aib residue for the turn-like geometry (Fig. 5(a)) Z-(Aib)4-OMe is remarkably sparse for a molecule this size,
observed in a 310-helix.
having only two competing structures within 10 kJ molÀ1 of the
The low-energy region of the energy level diagram for
Conformer B, which is 7.2 kJ molÀ1 above the global mini- global minimum. Both the global minimum and the second
mum, represents the first example of a C5/C7 bifurcated double lowest-energy (+3.5 kJ molÀ1) structures are F-F-10-10 structures
ring in which a single NH group interacts with the nearest- characteristic of a 310-helix, with very similar calculated IR
neighbor CQO groups on either side of it. We have already spectra that are nominally consistent with the observed spec-
noted that conformer C, the 5-5 structure, is the lowest (and only) trum of conformer A, whose S0–S1 origin dominates the R2PI
example of a fully-extended structure. Thus, all three observed spectrum. Conformer B is assigned to the structure 3.9 kJ molÀ1
conformers of Z-(Aib)2-OMe are the lowest-energy examples of above the global minimum, which is the lowest energy member of
three unique conformational families, suggesting that barriers the F-F-10-7 family. Above 10 kJ molÀ1, there are many structures
between members of the same family are relatively low, but inter- with similar energies belonging to a wide variety of H-bonding
family barriers are substantial enough that collisional cooling architectures, only one of which was observed experimentally, and
between them is incomplete. The large intensity of conformer A is assigned as conformer C.
in the R2PI spectrum relative to B and C is consistent with B and
Interestingly, the calculations predict a large number of
C being higher in energy, and therefore having smaller popula- structures, 411 kJ molÀ1 above the calculated global mini-
tions. Admittedly, other factors beyond population can affect mum, trapped in F-F-10-10 conformational minima (left-most
relative sizes of peaks in the R2PI spectrum (e.g., Franck–Condon column of Fig. 10(a)). A C10 backbone hydrogen bond between
activity, S1 lifetime, photoionization cross section). Once again, the carbonyl oxygen of residue ‘‘i’’ and the amide hydrogen of
the lack of spectroscopic evidence for conformers of the F-7 residue ‘‘i + 3’’ is the defining characteristic of a b-turn, for
and F-F conformational manifolds computed to have energies which there is a taxonomy including many variations of f and
lower than those of assigned conformers B and C suggests c backbone angles for residues i + 1 and i + 2.50 Conformer A of
that structures may have small barriers for isomerization to Z-(Aib)4-OMe exhibits ‘‘Type III’’ b-turns characteristic of 310-helices,
conformer A.
for which (f2 and f3 B À571, c2 and c3 B À301)50,51 The higher-
Fig. 10(a) presents an energy level diagram for Z-(Aib)4-OMe, energy members of the F-F-10-10 manifold exhibit other classes
the largest member of the present series of Aib homopeptides. of b-turn structures, differing in the dihedral angles along the
Once again, the structures have been grouped into columns based incipient 310-helix, and in the strength of the C10 H-bonds that
on conformational/hydrogen-bonding family type. The first three are formed.
Fig. 10 (a) Energy level diagram showing different structures of Z-(Aib)4-OMe grouped by hydrogen-bonding family. Energies (kJ molÀ1) are relative to
the calculated global minimum structure of Z-(Aib)4-OMe. (b) Energy level diagram comparing the assigned structures for Z-(Aib)4-OMe with their
counterparts in Z-(Gly)4-OMe and a selection of other low-energy Z-(Gly)4-OMe structures. See text for further discussion. Energies (kJ molÀ1) on the
left-hand column are relative to the calculated global minimum of Z-(Aib)4-OMe or Z-(Gly)4-OMe (as appropriate). Energies on the right-hand axis of
(b) are relative to the F-F-10-10-(S) structure of Z-(Gly)4-OMe.
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The classically defined b-turn structure does not proscribe the
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Conformer B is assigned as an F-F-10-7 structure. The F-F-10-7
f, c angles of the i + 3 residue participating in a b-turn. The two structural manifold emerges from the F-F-10-10 structures by
lowest-energy F-F-10-10 conformers we calculated for Z-(Aib)4-OMe distortions of the dihedral angles in the third Aib residue away
exhibit f, c angles for the C-terminal (i.e., fourth) residue that from their archetypal 310-helical values. There are nine struc-
belong to the 310-helical region of the Ramachandran map. tures within the first 25 kJ molÀ1 that belong to this F-F-10-7
The major difference between these two low energy structures is family. The lowest energy F-F-10-7 structure (i.e., conformer B) is
that the minimum energy structure inverts the f, c angles of its almost 10 kJ molÀ1 more stable than the next structure in this
C-terminal residue, while the structure 3.5 kJ molÀ1 higher in family. Just as in the F-F-10-10 structures, the other members of
energy does not. That is, the minimum energy structure exhibits the F-F-10-7 family can be accounted for with combinations
a change of handedness for its fourth residue, as compared to of ring flips, reversals of the Aib[3] Ramachandran angles, and
preceding three residues. This ‘‘helix reversal’’, known as a modifications of c4.
Schellman motif, is a common phenomenon at helix termini in
Conformer C is tentatively assigned to a compactly folded,
protein structures and in the crystal structures of Aib oligomers.52–55 mixed-helix structure containing three intramolecularly-hydrogen-
The structure assigned to conformer A, with an energy bonded NH stretch transitions and a free NH stretch. This con-
3.5 kJ molÀ1 above the calculated global minimum, maintains former appears near the top of Fig. 10(a), in a column depicting
its handedness along the whole length of the peptide backbone the energy levels of conformations exhibiting 3 or 4 intramolecular
(i.e., non-Schellman) and provides a slightly better spectroscopic fit hydrogen bonds. (The final two columns of Fig. 10(a) indicate
to experiment. The calculated dipole moments of the Schellman the energies of conformations found to contain one or two
and non-Schellman structures are nearly identical, differing by intramolecular hydrogen bonds.) The structure assigned to
only 0.07 D. A comparison of these two calculated spectra with conformer C, a 14/11-7-7-F conformation, is shown in Fig. 8(c).
experiment is included in the ESI† (Fig. S1).
While the fit between experiment and theory in Fig. 7 is reason-
Elsewhere in the diagram, Schellman/non-Schellman (S/NS) able, the assigned structure is 24 kJ molÀ1 above the global
structural pairs appear. For instance, the F-F-10-10 structure at minimum, and keeps this assignment tentative. The combi-
+17.5 kJ molÀ1 differs from the global minimum by changes in nation of laser desorption with rapid collisional cooling in the
the dihedrals about the first Aib residue (f1, c1). (The energetic supersonic expansion may be trapping a small portion of the
cost for this rearrangement is quite high.) Its NS counterpart is population in this conformational well. Assignment of such a
also observed, and appears at +20.5 kJ molÀ1, as anticipated.
high-energy structure, while certainly uncommon, is not unpre-
The conformational variations within the F-F-10-10 confor- cedented. In fact, Dean et al. made a similar argument to justify
mational manifold include deviations of f and c of Aib[1] and/or the assignment of a high energy conformation in their work on
Aib[4], and in the orientation of the phenyl ring of the Z-cap. Z-(Gly)3-OH.22
These differences and combinations there-of have been anno-
tated on the energy level diagram in Fig. 10(a).
B. Comparison of Z-(Aib)n with Z-(Gly)n
The orientation of the phenyl ring can also have a significant A natural point of comparison exists between the present set of
impact on the DFT-computed energy of a given structure. In Aib homopeptides and their corresponding glycine (Gly) counter-
Fig. 10(a), structures annotated with ‘‘RF’’ incorporate a ‘‘ring parts. One point of commonality between the two is that, unlike
flip’’ in which the phenyl ring is rotated by B1801 around the chiral amino acids, where formation of secondary structures such
C–C(H2)–O–C(QO) dihedral angle of the Z cap, as compared to as helices occurs with a particular handedness (e.g., L-amino
this dihedral’s value for the rings in the two lowest energy acids preferentially form right-handed helices).50,56,57 Aib and Gly
structures. Such a geometry change comes at an energy cost are both achiral, and therefore have allowed Ramachandran
of about 8 kJ molÀ1. The phenyl ring orientation may further angles in which (+f, +c) and (Àf, Àc) are equivalent. Perhaps
destabilize the structure by adopting an anti-configuration in more importantly, Gly, with its two hydrogens, is the most flexible
which the dihedral angle value falls between Æ1201 to Æ1801. of the amino acids, with allowed Ramachandran angles that span
This orientation raises the energy of a given structure by the largest range of any of the amino acids.58 By contrast, Aib,
approximately 12 kJ molÀ1. The isolated nature of these ring with its two methyl groups, shows much more restricted (f, c)
adjustments has been tested by manually changing the ring preferences localized around (+571, +301) and (À571,À301), and
orientation, reoptimizing the structure at the DFT level, and readily forms 310-helices in solution. Comparing the inherent
comparing the resultant energies. In these tests, it was observed conformational preferences of Aib and Gly homopeptides in the
that adjusting the ring in this way only impacted the zero-point gas phase gives unique insight regarding the effects of dimethyl
corrected energy of a particular structure and did not impact substitution, particularly in light of the accumulating macro-
the predicted spectrum to any significant extent.
dipole of the 310-helix that accompanies the spatial correlation
The remaining annotations, not explicitly discussed here, all of amide-group orientations.59
occur at the ends of the molecule and primarily involve atoms
The structure observed for conformer A of Z-Aib-OH is
that do not participate in intramolecular hydrogen bonding. By strikingly similar to the assigned conformations for Z-Gly-OH
considering these geometry rearrangements individually and in in both its UV and IR spectroscopy.22 The limited backbone
concert with each other, the large number of unique structures length of Z-X-OH, where X = Aib or Gly, provides very little
within the F-F-10-10 family can be understood.
conformational flexibility, and makes the extended C5 structure
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a highly favorable geometry. The similarity in the UV spectra
While a direct experimental comparison between Z-(Aib)4-OMe
between both molecules reflects their nearly identical phenyl and Z-(Gly)4-OMe is not currently available, it is possible to
ring environments. The frequencies of the OH and NH stretch calculate the relative energies of optimized structures for
oscillators also fall within a few cmÀ1 of each other, again as a Z-(Gly)4-OMe at the same level of theory, and thus gain insight
consequence of the similar structures. However, conformer B of to the changes in relative energy that accompany dimethyl sub-
Z-Aib-OH does adopt a distinct conformation. The increased stitution of glycine residues. In addition, in order to place these
steric impact of dual methyl sidechains causes a distortion of structures for Z-(Gly)4-OMe into a more appropriate context, a
the fully extended structure and results in a backbone geometry conformational search was performed on Z-(Gly)4-OMe, and a
with dihedral angles characteristic of the 310-helix. With only a set of the low energy structures discovered by the force field
single Aib residue, the nascent ability of the dimethylated level search was submitted to DFT geometry optimization
peptide to induce this geometry is observed in Z-Aib-OH. The and harmonic, vibrational frequency calculation. The results
analogous Gly-based structure does not experience this steric of these calculations are shown in the energy level diagram
burden, and the second, minor conformer observed in Z-Gly-OH in Fig. 10(b).
retains the same dihedral angles as the major conformer and
simply experiences a reorientation of its aromatic ring.22
Several important deductions follow. Perhaps most startling
is that the F-F-10-10 structure that is the emergent form of a
The data for Z-(Aib)4-OMe can be compared qualitatively to 310-helix and the global minimum in Z-(Aib)4-OMe is now more
Z-(Gly)3-OH, with the fourth amide group in Z-(Aib)4-OMe having than 18 kJ molÀ1 above the lowest energy structure calculated
its NH group substituted for OH in Z-(Gly)3-OH.22 Despite this from our cursory search of Z-(Gly)4-OMe. In contrast, the
nominal similarity, the dominant conformer of Z-(Aib)4-OMe, the minimum energy Z-(Gly)4-OMe structure is a 14-7-7-F structure,
F-F-10-10, 310-helix, has no low-energy counterpart in Z-(Gly)3-OH. with its three hydrogen bonds pointing in both the N - C
In order for the Gly-based molecule to form a second C10 H-bond terminus and C - N terminus directions, while those in the
the cis-OH in the COOH group would have to reconfigure to trans, 310-helix point only from C - N. It is worth emphasizing that
an energetically costly isomerization. Instead, the major conformer we have not done an exhaustive search to find the global mini-
A of Z-(Gly)3-OH has an 11-7-7-OHÁÁÁp hydrogen-bonding pattern mum structure in Z-(Gly)4-OMe, so the true minimum may be
that is surprisingly similar to the 14/11-7-7-F pattern tentatively even lower in energy than the 14-7-7-F structure. Nevertheless,
assigned to the minor conformer C in Z-(Aib)4-OMe. The OHÁÁÁp the 14-7-7-F structure is an incipient form of the 14/16-helix
hydrogen bond in conformer A of Z-(Gly)3-OH would have its observed in Z-(Gly)5-NHMe, which had a full H-bonding pattern
equivalent in an NHÁÁÁp H-bond in conformer C of Z-(Aib)4-OMe, of F-14-7-7-16-7 using the present labeling scheme. The 14-7-7-F
but formation of this p H-bond is blocked by the ester cap. Instead, structure already has elements of a mixed-helix in that the C14
the ester CQO[4] forms the second leg of a C14/C11 bifurcated ring constitutes an i - i + 3 H-bond, while the C7 rings are
double-ring with NH [1] in the Aib-based molecule.
i + 2 - i. The dipole moment of this structure is 5.2 D, while
One of the most striking results of the Z-(Gly)n studies occurred the dipole moment of the F-F-10-10 polyglycine structure is
in the largest members of the series, Z-(Gly)5-OH and Z-(Gly)5- 12 D, consistent with the previous deduction that polyglycines
NHMe. While polyglycine peptides are known to adopt PGI/PGII in the gas phase preferentially fold into structures that reduce
structures (extended structures having f, c dihedral angles of the dipole moment.
À1501, 1471 and À771, 1451, respectively) in solution,60 Dean et al.
Substitution of Gly for Aib also has dramatic effects on the
discovered that these molecules instead form 14/16 mixed helices relative energies of the observed conformers of Z-(Aib)4-OMe.
in the gas phase.22 Unlike a- or 310-helices, mixed helices incorpo- For instance, the energy ordering of the assigned F-F-10-10 and
rate H-bonds that include both N - C terminus and C - N F-F-10-7 structures switches upon substitution of Gly for Aib.
terminus H-bonds that reduce the magnitude of the dipole In the Aib-based structures, conformer B, which incorporates a
moment which frequently accompanies helix formation.22 In the C7 hydrogen bond instead of a second C10 (i.e., F-F-10-7), is
gas phase, the calculated energies of polyglycine PGII or 310-helices calculated to be approximately 4 kJ molÀ1 higher in energy than
increase relative to the 14/16 mixed helix as the helix length grows. the global minimum (F-F-10-10) structure. When Gly residues
Dean et al. postulated that the accumulating macrodipole of the compose the backbone instead, the F-F-10-7 structure becomes
polyglycine helices (PGII (9 D), 310 (21 D)) destabilizes them in the lower in energy than F-F-10-10(s) by B6 kJ molÀ1, a relative
non-polar environment of the gas phase relative to the low dipole energy shift of nearly 10 kJ molÀ1
.
moment of the mixed helix (m = 2.8 D in Z-(Gly)5-NHMe) leading to
the preference for mixed helical structures.
Even the high-energy 14-7-7-F structure tentatively assigned
to conformer C (Fig. 10(b)) of Z-(Aib)4-OMe undergoes a signi-
The present work on Aib homopeptides shows the reverse to ficant shift in relative energy upon replacement of all the Aib
be true in Z-(Aib)4-OMe. The F-F-10-10 structure, an emergent residues with glycine residues. This structure has an energy
form of the 310-helix, is preferred over all alternatives, despite 24 kJ molÀ1 above the global minimum for the Z-(Aib)4-OMe
its sizable macrodipole of 13.7 D. We surmise on this basis that case. When Gly residues are present instead, the significantly
dimethyl substitution at Ca restricts the energetically allowed decreased steric crowding from the side chains, lowers the energy
(f, c) angles about each Aib residue to such an extent that of the Gly version of conformer C by approximately 17 kJ molÀ1
310-helix formation is observed in spite of its significant (relative to the F-F-10-10 structure), while maintaining the same
macrodipole.
overall structure. (There is a slight backbone dihedral adjustment
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Table 2 Shown below are the structures, relative energies, H-bonding patterns, and relevant dihedral angles for Gly-based analogues of the assigned
structures for Z-(Aib)4-OMe as well as the selected structures from our cursory conformational search on Z-(Gly)4-OMe. Structures having a direct
Z-(Aib)4-OMe counterpart are listed as S and NS for Schellman and non-Schellman, B for conf. B, and C for conf. C. Non-analogous Z-(Gly)4-OMe
structures are simply numbered based upon their energy ordering. All angles are given in degrees
Relative energy
H-bonding
pattern
Ring
orientation
Molecule
(kJ molÀ1
)
f (1)
c (1)
f (2)
c (2)
f (3)
c (3)
f (4)
c (4)
Z-(Gly)4-OMe (S)
Z-(Gly)4-OMe (NS)
Z-(Gly)4-OMe (B)
Z-(Gly)4-OMe (C)
Z-(Gly)4-OMe #1
Z-(Gly)4-OMe #2
Z-(Gly)4-OMe #3
Z-(Gly)4-OMe #4
Z-(Gly)4-OMe #5
Z-(Gly)4-OMe #6
+18
+22
+13
+26
0
F-F-10-10 (S)
F-F-10-10 (NS)
F-F-10-7
14-7-7-F
14-7-7-F
F-11-7-7
14-7-7-F
11-7-7-F
11/14-7-7-p
F-11-7-7 (OMe)
67
66
62
85
99
À66
À65
À69
À90
À81
57
80
81
77
À55
À12
À16
À13
14
À69
À70
À73
80
À6
À5
À109
À96
80
À64
À93
81
121
148
127
À81
25
1
À74
150
2
À54
À23
À165
À161
54
69
À71
À104
53
14
À13
À176
41
171
À168
60
114
66
13
À19
À80
À67
69
66
83
137
+5
À85
À84
À101
À169
83
À143
À78
À78
À75
140
À89
À85
À83
À79
91
À122
À103
À86
À92
135
+11
+17
+22
+27
70
54
75
À70
that results in the breaking of the first C7 hydrogen bond.
These dihedral angles are shown in Table 2.)
The observant reader will point out that the global mini-
mum structure found by our cursory probe of the Z-(Gly)4-OMe
conformational space also adopts a 14-7-7-F geometry. Indeed,
this is yet another instance in which adjustments to the dihedral
angles along the backbone result in dramatic changes in energy.
Upon substitution of Gly residues for Aib residues, the dihedral
angles of conformer C change by no more than 201 at any given
position. In fact most of the dihedrals change by less than 101,
indicating that the reduction in energy likely comes primarily
from relief of the steric strain imposed by the methyl side chains.
Comparing the dihedral angles of the higher energy 14-F-7-F
(Gly-substituted Z-(Aib)4-OMe conformer C) to the minimum
energy 14-7-7-F structure of Z-(Gly)4-OMe, on the other hand,
reveals dihedral angles that differ to a much larger extent
(201–100+1) at each position. Therefore, we conclude that the
reduction in energy is associated with adoption of a geometry
that is inherently preferred by the backbone.
Fig. 11 Schellman (a) and non-Schellman (b) versions of conformer A
of Z-(Aib)4-OMe overlaid with previously determined crystallographic
structure for a Z-(Aib)4-OMe oligomer possessing a 310-helical structure
extracted from a unit cell. All hydrogen atoms have been removed to
better show the backbone overlap. Hydrogen-bonding interactions have
been indicated with dotted red lines.
Finally, there is one aspect of the energy level diagram that is dictate the structure of this molecule, and the crystal structure
relatively conserved within the Aib - Gly substitution; namely, is essentially unperturbed from the gas phase minimum. The
the relative energy difference between the Schellman and non- fact that the structure is stable regardless of its environment also
Schellman structures for conformer A, which remains near speaks to the strong local structural preferences induced in the
3 kJ molÀ1 between the two molecules. Thus, it appears the peptide backbone by the Aib residue, a rather remarkable result
energy requirement for flipping the C-terminal residue from given the small size of the methyl groups.
one handedness does not arise from the dispersive/London
One significant distortion is seen in the comparison between
forces of Aib’s methyl side chains, but instead may be due to the the non-Schellman structure and the crystal structure at the
different electrostatic interaction of the polar ester group with C-terminal end of the molecule. In the tentatively assigned (non-
the rest of the molecule. Indeed, the recent crystal structure of Schellman) gas-phase structure, the C-terminal Aib residue has
Z-(Aib)3-Gly-OtBu, which is terminated with a glycine residue, also dihedral angles that maintain the handedness of the helix, while in
exhibits the C-terminal helix reversal of the Schellman motif.61
the crystal structure the handedness of the final residue is flipped.
This difference is most clearly seen in the front view, shown
in Fig. 11(b). In the crystal phase, the Schellman motif enables
C. Comparison with crystal structures
Fig. 11 compares the calculated Schellman and non-Schellman formation of a pair of intermolecular NHÁÁÁCQO hydrogen bonds
structures for the F-F-10-10 conformer of Z-(Aib)4-OMe with an with each of two adjacent Aib oligomers within the unit cell. By
available X-ray crystal structure of the same molecule taken from aligning two 310-helical segments end to end, the free NH groups at
the work of Ranganathan et al.62 Along most of the peptide back- the N-terminus of one oligomer interact with the free CQO groups
bone, the overlay between the gas phase F-F-10-10 and the X-ray located at the C-terminus of the adjacent molecule. The isolated
crystal structure is excellent, indicating that even in the presence molecules probed by our experiment removes the possibility for
of close neighbors in the solid state, intramolecular forces intermolecular hydrogen bonding between Aib oligomers.
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While the vibrational spectrum shows a slightly better fit with Acknowledgements
the standard 310-helix form of F-F-10-10, both structures are low
in energy, with the Schellman version calculated to be the global
minimum. Since the difference between the calculated infrared
spectra for the two structures is subtle (see Fig. S1 in ESI†), if the
energy difference is real, it is likely that the experimentally
observed structure is the Schellman version, in which case the
correspondence between gas phase and crystal is near-perfect.
JRG, DMH, AOHC, KNB, and TSZ gratefully acknowledge sup-
port for this work from the National Science Foundation (NSF
CHE-1465028). MCR gratefully acknowledges support for this work
from the Lawrence Family Fellowship at Fairfield University.
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