Thermodynamic Scale of β-Amino Acid Residue Propensities for an α-Helix-like Conformation

Article abstract of DOI:10.1021/jacs.8b05162

A thiol-thioester exchange system has been used to measure the propensities of diverse β-amino acid residues to participate in an α-helix-like conformation. These measurements depend on formation of a parallel coiled-coil tertiary structure when two peptide segments become linked by thioester formation. One peptide segment contains a "guest" site that accommodates diverse β residues and is distal to the coiled-coil interface. We find that helix propensity is influenced by side chain placement within the β residue [β³ (side chain adjacent to nitrogen) slightly favored relative to β² (side chain adjacent to carbonyl)]. The previously recognized helix stabilization resulting from five-membered ring incorporation is quantified. These results are significant because so few quantitative thermodynamic measurements have been reported for α/β-peptide folding.

Full text of DOI:10.1021/jacs.8b05162

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Thermodynamic Scale of β‑Amino Acid Residue Propensities for an
α‑Helix-like Conformation
Brian F. Fisher,† Seong Ho Hong,‡ and Samuel H. Gellman*
Department of Chemistry, University of Wisconsin−Madison, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: A thiol-thioester exchange system has been
used to measure the propensities of diverse β-amino acid
residues to participate in an α-helix-like conformation.
These measurements depend on formation of a parallel
coiled-coil tertiary structure when two peptide segments
become linked by thioester formation. One peptide
Figure 1. Cartoon of thioester exchange experiment. X indicates
segment contains a “guest” site that accommodates
position of guest residue, the helix propensity of which can be
diverse β residues and is distal to the coiled-coil interface.
evaluated. “β” Indicates a β residue kept constant for all T peptides.
[TE−T]u and [TE−T]f indicate folded and unfolded states of the
coiled-coil-forming full-length thiodepsipeptide, respectively.
We find that helix propensity is influenced by side chain
placement within the β residue [β3 (side chain adjacent to
nitrogen) slightly favored relative to β2 (side chain
Although the cylinders shown for the T and TE segments imply
adjacent to carbonyl)]. The previously recognized helix
stabilization resulting from five-membered ring incorpo-
ration is quantified. These results are significant because
so few quantitative thermodynamic measurements have
been reported for α/β-peptide folding.
helicity, these segments undoubtedly explore both helical and
nonhelical conformations.
The experimental design is based on our recent strategy for
measuring α-helix propensities of α residues via thioester
exchange (Figure 2).7 Two helix-forming segments of 16−17
residues each are linked in parallel by a flexible segment that
contains a thioester bond. One segment (TE) provides the acyl
group, and the other (T) provides the thiol (Cys side chain)
for the thioester bond.8 Each peptide sequence features a
heptad repeat motif, with hydrophobic residues at positions a
and d of each repeat to encourage intramolecular coiled coil
formation. Adoption of an α-helix (or α-helix-like) con-
formation by each peptide segment is encouraged by the
placement of Arg and Glu residues at many positions other
than a and d to allow intrahelical salt bridges. Upon
intramolecular parallel coiled coil formation, Arg residues at
e and g positions of the TE segment make interhelical salt
bridges with Glu residues at e and g positions of the T segment.
When the small tyrosine-derived thiol Y is present in
addition to the TE−T thiodepsipeptide, a thiol-thioester
exchange reaction occurs. The equilibrium constant for the
folding process (Kfold), i.e., for interconversion between the
unfolded state of TE−T and the parallel coiled-coil tertiary
structure, can be determined from the thiol-thioester
equilibrium constant (Kex), which is measured via HPLC.
ΔGfold, calculated from Kfold, provides the basis for a propensity
scale. Control studies indicate that these ΔGfold values depend
on intramolecular coiled-coil interactions between the T and
TE segments, as required by our experimental design.9
he α-helix is the most common regular secondary
Tstructure within globular proteins, and this situation has
inspired many efforts to establish quantitative α-helix
propensity scales for the 20 proteinogenic residues.1
Polypeptides containing mixtures of α- and β-amino acid
residues, with up to 33% β residues evenly distributed along
the backbone, can adopt α-helix-like conformations.2 These α-
helix-mimetic α/β-peptides can engage binding sites on
proteins that evolved to recognize α-helical ligands, leading
to inhibition of specific protein−protein interactions or
activation of cell-surface receptors.3 The presence of β residues
blocks recognition by proteases and immune receptors, which
raises the prospect of biomedical utility.4 Bioactive α/β-
peptides have been generated via solid-phase synthesis to date,
but prospects for accessing such peptides via biosynthetic
machinery are steadily improving.5 In light of these develop-
ments, it would be valuable to establish a thermodynamic scale
for the propensities of β residues to reside in an α-helix-like
secondary structure; we report such a scale here.
Among α residues, variations in α-helix propensity arise
largely from variations in the side chain.6 There is a richer
landscape of possibilities among β residues because side chains
can be placed adjacent to nitrogen (β3 residues), adjacent to
carbonyl (β2 residues), or at both positions (β2,3 residues). β2,3
residues can contain rings, with variable size and stereo-
chemistry. To survey this range of β residue substitution
patterns, we turned to a propensity assessment strategy based
on thiol-thioester exchange (Figure 1).
Our previous study demonstrated that the identity of α
residues at f positions in the T segment influences ΔGfold as
expected based on established α-helix propensity scales, so
Received: June 1, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b05162
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
A first set of measurements evaluated variations in β3 residue
side chain (Figure 3). Removal of the methyl side chain, to
Figure 2. (a) Helix-wheel diagram of thioester peptide TE, with
fragment derived from small thiol Y highlighted in orange, and thiol
α/β-peptide T. Guest β residue position X is highlighted in red. (b)
Sequence of full-length thiodepsipeptide TE−T. Succ = succinyl. (c)
Structure of β3-hAla, at position 8′ in all T α/β-peptides, and general
structure of β residues tested at position 15′.
long as the “guest” residue side chain is not ionized.7 The good
correlation between our thermodynamic scale and α-helix
propensity scales derived by other methods supports our
design hypothesis that guest residues in f positions of the T
segment do not contact the TE segment in the helix−loop−
helix tertiary structure (folded state). These findings provide a
foundation for using the TE−T system to measure β residue
propensities to reside in an α-helix-like conformation. A first
step toward that goal, involving the T variant containing β3-
homoalanine (β3-hAla) at two f positions, indicated that a β3-
hAla residue within an α-helix-like conformation is 0.6 kcal/
mol less favorable than an Ala residue within an α-helix.7
The new studies employ the version of T containing β3-hAla
at two f positions as the point of reference. We monitor
changes in ΔGfold as the β residue is altered at just one
position; the other f position remains β3-hAla. This design
isolates the impact of altering β residue substitution at a single
site within a constant α/β backbone. For the reference peptide,
with β3-hAla at the “guest” position, ΔGfold = −0.87 kcal/mol.7
A critical test of our design hypothesis involves the derivative
of this reference peptide with Leu13′ → Asn, for which ΔGfold
= +0.17 kcal/mol. This d position should participate in the
coiled-coil interface, and Leu → Asn should therefore
substantially diminish ΔGfold if folding depends upon intra-
molecular coiled coil formation, as intended. The large
destabilization that we observe to result from the Leu13′ →
Asn modification supports our assumption that a coiled coil-
like association is retained when β residues are placed at f
positions in the T segment, and that intramolecular
interactions between T and TE segments involve helical
conformations of both segments.
Figure 3. (a) Thioester exchange-derived ΔGfold values for α/β-
peptides containing a β3- or β2-homoamino acid guest residue. (b)
Structures of β3- and β2-homoamino acids.
generate β-hGly, was slightly destabilizing (Table 1).7
Enlarging the side chain, by replacing β3-hAla with either β3-
hLeu or β3-hVal, seemed to cause a slight enhancement in
helical propensity. The thermodynamic similarity we observe
for β3-hLeu and β3-hVal in an α-helix-like conformation (Table
1) contrasts with the trend in proteins, where Leu has a
significantly higher α-helix propensity than does Val.1 In
general, side chain branching adjacent to the backbone is
unfavorable for an α-helix, but this trend is not retained for β
residues in an α-helix-like conformation. β3-hGlu appears to
display a significantly higher helix propensity relative to β3-
hAla, behavior we attribute to favorable intrahelical Coulombic
interactions of the anionic β3-hGlu side chain with spatially
proximal cationic Arg side chains in the folded state (positions
b and c).10 A similar effect was observed for Glu in the guest
sites of the analogous α-helical system.7
All β3 residues mentioned so far are enantiospecifically
derived from the corresponding L-α-amino acids (S config-
uration) with retention of configuration. Inversion of
configuration (ent-β3-hAla) leads to a decline in helix
propensity of ≈ 0.7 kcal/mol, which is qualitatively consistent
with but smaller than the decline in α-helix propensity
reported for D-Ala (R configuration) relative to L-Ala.1b,7 The
B
DOI: 10.1021/jacs.8b05162
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Journal of the American Chemical Society
Communication
Table 1. ΔGfold Measured by Thioester Exchange for Thiol
a
Peptides Containing β-Amino Acid Guest Residues
b
β Residue class
Guest residue
ΔGfold (kcal/mol)
β3
β-hGly
β3-hAla
β3-hAla(Asn)
−0.63 0.05
−0.87 0.15
0.17 0.15
c
β3-hLeu
β3-hVal
−1.05 0.13
−1.06 0.04
−1.30 0.05
−0.17 0.03
−0.39 0.06
−0.60 0.10
−0.18 0.12
−0.69 0.05
−0.22 0.09
−1.40 0.08
−0.18 0.06
−0.50 0.04
−0.11 0.07
−0.51 0.02
−0.97 0.03
−1.30 0.04
−1.23 0.07
−1.81 0.08
β3-hGlu
ent-β3-hAla
β
3,3-hAib
β2
β2-hAla
ent-β2-hAla
β2-hVal
β
2,2-hAib
β2,3
ACPC
c
ACPC(Asn)
β
2,3-hAla
ent-ACPC
(2R,3S)-ACPC
ACHC
ATC
gAPC
Figure 4. (a) Thioester exchange-derived ΔGfold values for α/β-
peptides containing a β2,3-disubstituted β-amino acid guest residue.
Horizontal line represents ΔGfold for β3-hAla. (b) Structures of β2,3-
disubstituted β-amino acids.
spAPC
a
ΔGfold = −RT ln(Kfold). Guest residue position within T is defined in
Figure 2. Conditions: 50 μM each TE and T, 50 mM phosphate, pH
7.0, 2 mM TCEP, room temperature. Mean standard deviation of
at least five experiments. Leu → Asn substitution at coiled coil
b
Ala with β3-hAla leads to a 0.6 kcal/mol decline in helix
stability,7 our findings indicate that replacing β3-hAla with
(S,S)-ACPC largely restores helix stability to the pure α
backbone level.
c
interfacial residue 13′ of T.
The (S,S)-β2,3-dimethyl residue β2,3-hAla, an acyclic
analogue of (S,S)-ACPC, has a helix propensity ≈0.9 kcal/
mol less favorable than that of (S,S)-ACPC. This observation
may reflect unfavorable gauche interactions between the
methyl side chains in the helical conformation. (R,R)-ACPC
displays a much lower helix propensity relative to (S,S)-ACPC
(ΔΔG ≈ 1.3 kcal/mol), as expected for a peptide composed
largely of L-α residues. cis-(2R,3S)-ACPC also has a low helix
propensity, ≈0.9 kcal/mol less favorable relative to (S,S)-
ACPC. Six-membered ring constraint, as in (S,S)-trans-2-
aminocyclohexanecarboxylic acid (ACHC), leads to a ≈0.4
kcal/mol decrease in helix propensity relative to the five-
membered ring constraint in ACPC.
Several analogues of (S,S)-ACPC bearing polar functionality
in the ring were evaluated. The neutral ATC residue16
(tetrahydrofuran ring) manifests a propensity similar to that
of ACPC, as does an analogue bearing a positive charge
(gAPC).17 In contrast, an analogue bearing a negative charge
(spAPC)18 displays a substantially higher propensity. The high
helix propensity observed for the anionic spAPC residue
parallels the high propensity observed for β3-hGlu as guest; we
propose that favorable intrahelical Coulombic interactions with
proximal cationic side chains (Arg) in the folded state explain
this trend. The apparent lack of unfavorable Coulombic
interactions with the cationic gAPC side chain might reflect the
fact that the position of the charge is tightly constrained in this
case.
Aib residue (gem-dimethyl substitution) has an α-helix
propensity larger than that of Ala,1b,11 but β3,3-hAib manifests
a significantly lower helix propensity relative to β3-hAla.
We evaluated β2 residues, in which the side chain position is
shifted relative to β3 residues. The S configuration of β2-hAla is
thermodynamically preferred relative to the R configuration in
the guest position, which supports previous assumptions.12
The difference in helical propensity between the two
configurations, ≈0.4 kcal/mol, is smaller than for the two
configurations of β3-hAla.
Our results suggest a small decline in helix propensity for β2
residues relative to β3 isomers (β-hAla and β-hVal, Table 1).
No significant difference could be detected between the helix
propensities of β2 and β3 residues in the only previous
quantitative comparison, which involved solvent-exposed
helical sites within a tertiary structure.12
Side chain branching adjacent to the backbone has little
impact on the helix propensity of β2 residues (β2-hAla vs β2-
hVal), which parallels the trend among β3 residues. The lower
helix propensity of β2,2-hAib relative to β2-hAla represents
another parallel with the β3 series.
Previous comparisons have suggested that cyclic β residues
with a five-membered ring constraint manifest enhanced helix
propensity relative to β3 residues.2,13−15 (S,S)-Trans-2-amino-
cyclopentanecarboxylic acid (ACPC) exemplifies these helix-
prone β-amino acids. We observe ≈0.5 kcal/mol enhancement
in helix propensity for ACPC relative to β3-hAla (Figure 4). In
a critical control experiment, we find that Leu13′ → Asn leads
to ≈1.2 kcal/mol destabilization with ACPC at the guest site,
which suggests that the intended coiled coil-like tertiary
packing between the TE and T segments is maintained when a
cyclic β residue is placed at the guest position. Since replacing
Quantitative information on helix propensities for β-amino
acid residues has been very limited to date,13 and there is no
precedent for the range of substitution patterns that we have
evaluated here. Circular dichroism signatures of the α/β-
peptide helices described above are qualitatively consistent
with the thioester exchange data.9
C
DOI: 10.1021/jacs.8b05162
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Journal of the American Chemical Society
Communication
̈
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Structural mimicry has been documented also at lower
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The backbone torsion angles of the β residues are relatively
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variation in α:β proportion and distribution. We therefore
expect that the trends in β residue propensity reported here
will be generally applicable to α/β-peptides that adopt α-helix-
like conformations; however, additional studies will be
required to test this prediction. α-Helix-mimetic α/β-peptides
display properties of considerable interest from a biomedical
perspective,2−4 and the thermodynamic data presented here
should support efforts to refine α/β-peptide candidates for
specific applications.
(7) Fisher, B. F.; Hong, S. H.; Gellman, S. H. J. Am. Chem. Soc. 2017,
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(8) Steinkruger, J. D.; Woolfson, D. N.; Gellman, S. H. J. Am. Chem.
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(9) See Supporting Information.
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84, 8898.
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P.; Fontana, A. Biochemistry 1998, 37, 1686.
(12) Tavenor, N. A.; Reinert, Z. E.; Lengyel, G. A.; Griffith, B. D.;
Horne, W. S. Chem. Commun. 2016, 52, 3789.
(13) Price, J. L.; Hadley, E. B.; Steinkruger, J. D.; Gellman, S. H.
Angew. Chem., Int. Ed. 2010, 49, 368.
(14) Reinert, Z. E.; Horne, W. S. Chem. Sci. 2014, 5, 3325.
(15) Kreitler, D. F.; Mortenson, D. E.; Forest, K. T.; Gellman, S. H.
J. Am. Chem. Soc. 2016, 138, 6498.
(16) Imamura, Y.; Umezawa, N.; Osawa, S.; Shimada, N.; Higo, T.;
Yokoshima, S.; Fukuyama, T.; Iwatsubo, T.; Kato, N.; Tomita, T.;
Higuchi, T. J. Med. Chem. 2013, 56, 1443.
(17) Demizu, Y.; Oba, M.; Okitsu, K.; Yamashita, H.; Misawa, T.;
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b05162.
■
S
Experimental details including amino acid synthesis data,
peptide synthesis procedures, thioester exchange UPLC
chromatograms (PDF)
AUTHOR INFORMATION
Corresponding Author
*gellman@chem.wisc.edu
■
(18) Lee, H. S.; Syud, F. A.; Wang, X.; Gellman, S. H. J. Am. Chem.
Soc. 2001, 123, 7721.
ORCID
Brian F. Fisher: 0000-0003-1727-2494
Present Addresses
†Brian F. Fisher, Department of Chemistry, Indiana University,
Bloomington, Indiana 47405
‡Seong Ho Hong, Department of Chemistry, New York
University, New York, New York 10003
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the National Science
Foundation (CHE-1565810). The authors thank Dr. Young-
Hee Shin for providing small thiol Y and Dr. Dale F. Kreitler
for providing Fmoc-protected ATC.
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DOI: 10.1021/jacs.8b05162
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