C12 helices in long hybrid (αγ)n peptides composed entirely of unconstrained residues with proteinogenic side chains

Article abstract of DOI:10.1021/ol500307p

Unconstrained γ⁴ amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (αγ)_n sequences. The C₁₂ helical conformation for the decapeptide, Boc-[Leu-γ⁴(R)Val

Full text of DOI:10.1021/ol500307p

Letter
pubs.acs.org/OrgLett
C12 Helices in Long Hybrid (αγ)n Peptides Composed Entirely of
Unconstrained Residues with Proteinogenic Side Chains
Rajesh Sonti,‡,†,∥ Bhimareddy Dinesh,†,∥ Krishnayan Basuroy,§ Srinivasarao Raghothama,‡
Narayanaswamy Shamala,§ and Padmanabhan Balaram*,†
†Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, 560012
‡NMR Research Centre, Indian Institute of Science, Bangalore, India, 560012
§Department of Physics, Indian Institute of Science, Bangalore, India, 560012
S
* Supporting Information
ABSTRACT: Unconstrained γ4 amino acid residues derived by homologation of proteinogenic
amino acids facilitate helical folding in hybrid (αγ)n sequences. The C12 helical conformation for
the decapeptide, Boc-[Leu-γ4(R)Val]5-OMe, is established in crystals by X-ray diffraction. A
regular C12 helix is demonstrated by NMR studies of the 18 residue peptide, Boc-[Leu-
γ4(R)Val]9-OMe, and a designed 16 residue (αγ)n peptide, incorporating variable side chains.
Unconstrained (αγ)n peptides show an unexpectedly high propensity for helical folding in long
polypeptide sequences.
expanded analogues of the 310 and α-helical structures in α-
omologated amino acid residues in which additional
Hmethylene groups have been inserted into the backbone polypeptides and proteins.10 Thus far, definitive character-
ization of hybrid helical structures has been achieved in
designed peptides containing conformationally constrained
residues, which are anticipated to promote local folding. For
example, the C12 helix in αγn sequences has been established in
crystals of (UγV)n (U = Aib; γV = γ4(R)Val) sequences ranging
in length up to 16 residues.11 Figure 1a illustrates the
conformation in crystals of Boc-(UγV)5-OMe in which the
hydrogen bonds of a successive C12 (4→1 COi---HNi+3)
stabilize the folded backbone resulting in an expanded analogue
of the 310 helix. C12 helices have also been characterized in Aib-
γ4Phe sequences,8,12 Gabapentin (Gpn) containing sequen-
ces,13 and multiply substituted preorganized residues.14 The βγ
C13 helix, which is a backbone expanded analogue of the α-
helix, has recently been described in sequences containing
constrained β- and γ-residues.7c,13d The intriguing observation
of two successive C12 hydrogen bonded αγ turns (incipient C12
helix) in short unconstrained αγ peptides,15 together with the
observation that γ- peptides of the type (γVal)n,(γLeu)n, and
(γIle)n (n = 4−10) yield C14 helices,9 suggested a high intrinsic
propensity of γ4(R) substituted residues to adopt locally folded
gauche (g+) conformations about the Cγ−Cβ (θ1) and Cβ−Cα
(θ2) bonds. Encouraged by these results, we examined the
possibility of designing hybrid sequences composed entirely of
α- and γ-residues varying the side chains present, in order to
have shown a surprising tendency to promote folded
polypeptide conformations, despite the introduction of addi-
tional degrees of torsional freedom.1 The foldability of γ-
peptides derived from homologues of the proteinogenic α-
amino acids was first reported, in the studies of short peptides
in organic solvents, by Seebach2 and Hanessian3 in the 1990s.
These observations coincided with the discovery by Gellman
that oligo-β-peptides form novel helical structures, unambigu-
ously characterized by single crystal X-ray diffraction.4 The
rapidly advancing field of foldamers,1b,5 which focuses on non-
α-amino acid backbones, has stimulated a great deal of interest
in the structural and functional properties of β- and γ-
peptides.1a,6 The definitive characterization of the precise
conformational features of β- and γ-peptides has been greatly
facilitated by the use of conformationally constrained
(“preorganized”) residues, which promote folded structures in
solution and enhance peptide crystallizability.7 Recent work on
γ-peptide sequences containing exclusively unconstrained
residues derived from the protein amino acids have established
a very high propensity for the formation of folded helical
structures,8 specifically the C14 helix in oligomeric γ-peptide
sequences.9
Hybrid polypeptides, which contain α-, β-, and γ-residues,
provide a means of expanding the structure space of synthetic
polypeptides, a desirable goal in the design of peptidomimetics.
Regular hybrid sequences (αβ)n, (αγ)n, (βγ)n, etc. afford an
opportunity to characterize helical structures, which are
Received: January 28, 2014
Published: March 3, 2014
© 2014 American Chemical Society
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OMe,11a clearly demonstrating that, despite replacement of the
conformationally constrained Aib residue by the unconstrained
Leu residue, a stable C12 helix is indeed formed.
Persistence of the C12 helix in the unconstrained (αγ)n
sequence is demonstrated by the solvent dependence of NH
chemical shifts in CDCl3/DMSO mixtures. Only two NH
γ
groups assigned to the N-terminus residues Leu1 and Val2
show appreciable downfield shifts upon increasing DMSO
concentration, indicative of their solvent exposure. The
remaining 8 NH groups show little or no dependence of
chemical shifts (Supplementary Figure S4b), suggesting that the
8 intramolecuar CO---HN hydrogen bonds anticipated in the
C12 helix are indeed observed in solution. The partial ROESY
spectrum shown in Figure 2 establishes the presence of NHi↔
Figure 1. Molecular conformation in crystals of (a) Boc-[Aib-
γ4(R)Val]5-OMe11 and (b) Boc-[Leu-γ4(R)Val]5-OMe(1). Note the
presence of two methanol molecules disrupting backbone hydrogen
bonding. (c) Solvation of the backbone atoms of peptide 1 is shown.
N---O (O--O) distances (Å) are indicated as broken lines. (d)
Sequences of peptides studied.
assess their ability to fold into stable helices. The growing
interest in the design of helical peptidomimetics,6a,16 for
disrupting protein−protein interactions,17 provides a strong
rationale for the detailed analysis of secondary structures in
hybrid polypeptides containing amino acids with proteinogenic
side chains. This report provides a definitive characterization of
long C12 helices in both the solid state and solution in αγn
sequences containing exclusively conformationally uncon-
strained residues, derived from proteinogenic amino acids. All
γ-residues are derived by homologation of the corresponding L-
(S)-α amino acids as specified by the Cahn−Ingold−Prelog
convention. For Val/Leu/Phe, homologation results in the R
configuration, while the specified configuration for Ala is S.1a
The choice of apolar hydrocarbon side chains facilitates
dissolution in organic solvents. Of the six sequences examined,
diffraction quality single crystals were obtained, thus far, only
for Boc-[Leu-γ4(R)Val]5-OMe (1). X-ray diffraction data are
summarized in Supplementary Table ST1, and coordinates are
deposited in the CCDC database (922799). Figure 1b shows a
view of the molecular conformation of 1 in crystals.
Supplementary Tables ST2 and ST3 list relevant backbone
torsional angles and hydrogen bond parameters.
Peptide 1 folds into a right handed αγ C12 helix stabilized by
six intramolecular 4→1 hydrogen bonds. Solvation of the
backbone at two potential hydrogen bonding sites by methanol
results in the disruption of two of the anticipated 4→1
hydrogen bonds in an ideal helix. Figure 1c shows a view of the
solvent invasion of the helix backbone, a feature that has also
been observed in helices formed in α-peptide crystals.18 The
average values of the backbone torsional angles (deg) at both α
(ϕ, ψ) and γ (ϕ, θ1, θ2, and ψ) residues for the C12 helix are as
follows: α, −79 14.5, −23.2 13.0; γ, −115.8 10.1, 54.4
7.0, 65.7 2.9, −134.5 10.8. These values correspond very
closely to that determined earlier for Boc-[Aib-γ4(R)Val]5-
Figure 2. Partial ROESY spectrum in CDCl3 of Boc-[Leu-γ4(R)Val]5-
OMe highlighting the dNN NOEs.
NHi+1 (dNN) NOEs at all five α-residues. The dNN distance of
approximately 3.6 Å across γ-residues results in unobservable
NOEs, precluding a reliable structure calculation using
experimental distance constraints. Encouraged by the evidence
for a well folded structure in peptide 1, we examined the 18
residue sequence, Boc-[Leu-γ4(R)Val]9-OMe (2). Figure 3a
illustrates well dispersed amide NH resonances in the 18
residue peptide. Sequence specific assignments could be
derived for residues 1−10, while resonance overlap precluded
complete assignment of the C-terminus residues. The solvent
titration curves shown in Figure 3b establish that, as in the case
of peptide 1, only the NH resonances of the two residues at the
N-terminus show a significant solvent dependence. These
results suggest that lengthening of the hybrid αγ sequence from
10 to 18 residues results in a propagation of the folded
structure over the entire length of the polypeptide chain. Local
helical conformations at the α-residues are also evident in the
observation of sequential dNN NOEs (Figure 3c). Stimulated by
the intrinsic ability of the unconstrained αγ sequences to form
well folded helical structures in solution, we turned to
sequences with greater side chain variability in order to
enhance the chemical shift dispersion of CαH and CγH
resonances, which should enable determination of multiple
distance constraints, permitting calculation of structures from
solution NMR data.14 Examination of NMR data for
octapeptides 3, 4, and 5 immediately revealed that enhanced
chemical shift dispersion was indeed observed, facilitating the
observation of additional NOEs between backbone protons of
the type CαH: CαHi↔ NHi+1, CαHi↔ NHi+2, CαHi↔ NHi+3
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1
Figure 3. Peptide 2: (a) Partial H NMR spectrum at 700 MHz in
CDCl3 illustrating NH resonance assignments (overlapping assign-
ments are marked with an asterisk; (b) delineation of hydrogen
bonded NH groups monitored by changes in chemical shifts of NH
protons with DMSO concentration; and (c) partial ROESY spectrum
highlighting the dNN NOEs.
1
Figure 4. Peptide 6: (a) Partial H NMR spectrum at 700 MHz in
CDCl3 highlighting the dispersion of NH resonances. (b) Change in
NH chemical shifts with DMSO concentration in a CDCl3-DMSO
mixture. (c,d) Partial overlay of TOCSY (blue), ROESY(red) spectra
highlighting CγHi↔CαHi+2 and CγH/CαH ↔ NH NOEs.
and CγH: CγHi↔ NHi+1, CγHi↔ NHi+2, CγHi↔ CαHi+2, CγHi↔
CβHi+2. The results of the NMR analysis of peptides 3−5 are
provided as Supporting Information (S5−S14 and ST6−
ST13). Figure 4 shows the results of NMR studies on the 16
residue peptide Boc-[Leu-γ4(R)Val-Phe-γ4(R)Leu-Ala-
γ4(R)Phe-Val-γ4(S)Ala]2-OMe (6). In this sequence four
different kinds of α- and γ-residues have been used and an
corresponding to 4→1 CO---HN (C12) hydrogen bonds in the
NMR derived structure are 3.2−3.6 Å. A feature of the αγ C12
helix is the large number of nonsequential backbone
interproton distances, which fall within the NOE observable
limits. These diagnostic distances involving the CαHi/CγHi
protons are schematically illustrated in Figure 5b and 5c. The
large number of restraints derived from backbone proton NOEs
provides a robust structural characterization of the αγ C12 helix
in solution.
The results presented above describe the crystallographic
characterization of the longest C12 helix in an αγ hybrid peptide
sequence composed entirely of unconstrained residues reported
thus far. The remarkable stability of the αγ C12 helix even in the
absence of helix nucleating, “preorganized” residues is
established by the NMR structure determination of the
continuous helical conformation in a 16 residue (αγ)n peptide
(6). Taken together, with a previously reported characterization
of C14 helices in unconstrained γn sequences,9 the results
establish a very high propensity of γ4 residues derived by
homologation of proteinogenic amino acids to facilitate helical
folding in oligopeptide sequences. These observations should
promote the rational design of protein mimics with hybrid
peptide backbones.19
1
extremely well dispersed H NMR spectrum was obtained at
700 MHz, permitting sequence specific assignments. Figure 4a
shows the dispersion of amide NH resonances, and Figure 4b
summarizes the dependence of chemical shifts in CDCl3/
DMSO solvent titration experiments. Only two N-terminus
NH resonances exhibit behavior characteristic of solvent
exposed NH groups establishing the involvement of the
remaining 14 NH groups in intramolecular hydrogen bonds.
The sequential dNN connectivities at the α-residues are
illustrated in Supplementary Figure S15, while Figure 4c and
4d show nonsequential backbone proton NOEs characteristic
of an αγ C12 helix. The key NOEs which provide important
constraints for distance geometry calculations are as follows:
NHαi↔ NHγi+1, CαHi↔ NHi+1, CαHi↔ NHi+2, CαHi↔ NHi+3,
CγHi↔ NHi+1, CγHi↔ NHi+2, and CγHi↔ CαHi+2. Using a set
of 57 distance constraints and 16 torsional angle constraints at
the α (3JC H−NH, 2.5−3.5 Hz) and γ (3JC H−NH, 8.6−10.5 Hz)
residues, an NMR structure calculation yielded the family of
structures shown in Figure 5a. The measured N−O distances
α
γ
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ASSOCIATED CONTENT
* Supporting Information
■
S
(12) Bandyopadhyay, A.; Jadhav, S. V.; Gopi, H. N. Chem. Commun.
2012, 48, 7170−7172.
Synthetic procedure, X-ray diffraction data, NMR parameters,
DMSO titration curves, relevant ROESY spectra, restraints used
in structure calculation, and dihedral angles are given. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail: pb@mbu.iisc.ernet.in.
Author Contributions
∥These authors contributed equally.
Notes
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Proteins 1990, 7, 62−73. (b) Karle, I. L.; Flippen-Anderson, J.; Uma,
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
R.S. acknowledges IISc for a fellowship. This research was
supported by a DBT-IISc partnership program grant.
■
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