Peptide α/310-helix dimorphism in the crystal state

Article abstract of DOI:10.1021/ja076656a

The fully C^α-methylated homo-peptide Ac-[l-(αMe)Val]₇-NHiPr is completely 3₁₀-helical when its crystals are grown from a methanol solution, whereas it is α-helical when crystallized from HFIP (1,1,1,3,3,3-hexafluoropropan-

Full text of DOI:10.1021/ja076656a

Published on Web 11/21/2007
Peptide r/310-Helix Dimorphism in the Crystal State
Marco Crisma,*,† Michele Saviano,‡ Alessandro Moretto,† Quirinus B. Broxterman,§
Bernard Kaptein,§ and Claudio Toniolo†
Institute of Biomolecular Chemistry, PadoVa Unit, CNR, Department of Chemistry, UniVersity of PadoVa, 35131
PadoVa, Italy, Institute of Biostructures and Bioimaging, CNR, 80134 Naples, Italy, and DSM Pharmaceutical
Products, AdVanced Synthesis, Catalysis and DeVelopment, MD 6160 Geleen, The Netherlands
Received September 4, 2007; E-mail: marco.crisma@unipd.it
The two helical structures most frequently found in peptides and
proteins, the R- and the 310-helix, can be visualized as the
successions of CdO‚‚‚H-N intramolecularly H-bonded pseudocy-
clic structures, the i r i+4 and i r i+3 forms, respectively.1 In
these two helices, also the number of residues per turn, the pitch,
and the φ,ψ backbone torsion angles are different, although not
dramatically. About 10% of all helical residues in globular proteins
are 310-helical.2 The 310-helices are typically short (3-4 residues)
and observed at the N- or C-terminus of R-helices. The 310-helical
structure has been suggested as an intermediate in R-helix folding
and melting processes.3 However, the 310-helix has been authen-
ticated at atomic resolution mainly in model peptides based on CR-
tetrasubstituted R-amino acids. In particular, the NR-acylated homo-
oligomers from R-aminoisobutyric acid -(Aib)11- and -(Aib)10-
represent the longest 310-helical peptide sequences so far investi-
gated by X-ray diffraction.4 Although transitions from R-helix to
random coil or to â-sheet structure have been extensively investi-
gated, only limited attention has been paid to the transition between
R- and 310-helices, despite the fact that this could form the first
step toward a molecular switch based on these two conformational
states. By comparing different, but related, peptides it has been
experimentally shown that the factors involved in shifting the
conformational preference from 310- to R-helix include the decreas-
ing percentage of CR-tetrasubstituted R-amino acids in the sequence
and the increasing length of the peptide.5 More subtle influence
can also be exerted by the amino acid sequence and the nature of
the terminal protecting (blocking) groups. Moreover, by spectro-
scopically analyzing the same peptide, based on a combination of
CR-tetrasubstituted and protein (CR-trisubstituted) R-amino acids,
under different experimental conditions (solvent, temperature), a
fast and reversible transition between the R- and 310-helical states
was observed, although in a very limited number of extremely
different cases.6
solution a slow, unexpected, acidolysis of the tert-butyl ester
functionality does take place, irreVersibly affording the correspond-
ing octapeptide free acid, which in turn rapidly folds into the R-helix
conformation, possibly due to the increased (by one) number of
H-bonding donors in its sequence.8c,d
On the basis of the above observations, in the present study we
focused on an N-acylated, chemically stable, L-(RMe)Val homopep-
tide with the same number of H-bonding donor NH groups as the
unstable tert-butyl ester described above, namely the NR-acylated
homoheptapeptide alkylamide Ac-[L-(RMe)Val]7-NHiPr, where Ac
is acetyl and NHiPr is isopropylamino (Supporting Information).
Circular dichroism (CD) experiments on Ac-[L-(RMe)Val]7-
NHiPr clearly showed that it undergoes a fast, solvent-driven,
reVersible R-helix/310-helix equilibrium, thus behaving as a mo-
lecular spring. More specifically, according to the CD patterns9
this peptide is overwhelmingly folded in the R-helix conformation
in HFIP solution, whereas it essentially adopts the 310-helix
conformation in the less polar methanol (MeOH) solution. Repeated
cycles of helix-to-helix conversion can be carried out, highlighting
inter alia the chemical stability of the peptide under the experimental
conditions used in this work (Supporting Information).
The X-ray diffraction structure of an impressive number of
oligopeptides containing CR-tetrasubstituted R-amino acids, from
five to twenty residues in length, have been solved and found to
be either fully developed 310- or R-helical, or mixed 310/R-helical.
This Communication describes in detail an example of an unam-
biguous R/310-helix dimorphism in an NR-acylated heptapeptide
amide, Ac-[L-(RMe)Val]7-NHiPr, crystallized from two different
solvents. All of the numerous homopeptides based on CR-tetrasub-
stituted R-amino acids, the 3D-structure of which have been solved
so far by X-ray diffraction (except the terminally protected, R-helical
octapeptide reported by Tanaka, Suemune, and their co-workers)10
have been found to adopt the 310-helical structure in the crystal
state.
The conformations of the three independent molecules (A, B,
and C) in the asymmetric unit of the unsolvated Ac-[L-(RMe)Val]7-
NHiPr crystallized from MeOH solution, where it is 310-helical, 1,
are very similar in that all are regular, right-handed, 310-helices
spanning the entire sequence (Figure 1). The average φ,ψ backbone
torsion angles for the seven residues are -55.2°, -30.3° (molecule
A), -55.3°,-33.5° (molecule B), and -54.9°,-34.3° (molecule
C), very close to those typical for a peptide 310-helix.1a In each of
the three peptide molecules all six, consecutive, i r i+3 (peptide)
CdO‚‚‚H-N (peptide or amide) intramolecular H-bonds are of
normal strength for these types of interactions, the range of O‚‚‚N
distances being 2.910(9)-3.258(9) Å.11 The major conformational
differences among the three molecules are seen in the ø1,1, ø1,2 side-
chain torsion angles of the seven L-(RMe)Val residues. While three
sets of angles are the same in the three molecules (t,g- at positions
Sometime ago, we decided to investigate systematically the
equilibrium between R- and 310-helices beginning from simplified
peptide sequences formed exclusiVely by the same CR-tetrasubsti-
tuted R-amino acid. From our previous studies it was already known
that among the chiral residues of this class the â-branched CR-
methyl-L-valine [L-(RMe)Val] is that with the most pronounced bias
toward the right-handed 310-helix.5b,7 Therefore, our first target in
this area was an N-acylated L-(RMe)Val homo-octapeptide tert-
butyl ester which was shown to undergo an intriguing phenomenon,
namely a slow and irreVersible conversion from 310- to R-helix,
the rate of which is particularly enhanced by high solvent polarity
(e.g., in 1,1,1,3,3,3-hexafluoropropan-2-ol, HFIP).8a,b However,
more recently we have unambiguously demonstrated that in HFIP
† University of Padova.
‡ Institute of Biostructures and Bioimaging.
§ DSM Pharmaceutical Products.
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J. AM. CHEM. SOC. 2007, 129, 15471-15473
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C O M M U N I C A T I O N S
Figure 2. X-ray diffraction structures of the two independent peptide
molecules (A and B) in the asymmetric unit of Ac-[L-(RMe)Val]7-NHiPr
bis-HFIP solvate (2) (crystals grown from an HFIP solution). H-atoms have
been omitted for clarity. Dashed lines represent intramolecular CdO‚‚‚
H-N H-bonds.
surprising as it is characteristic of a large number of R-helices in
peptides and globular proteins.1,2,5
Figure 3 shows the interactions between the cocrystallized HFIP
molecules and the three C-terminal carbonyls of each of the peptide
molecules A and B. The details of H-bonding are not strictly
equivalent. In both complexes the -OH group of one HFIP
molecule is H-bonded to the C-terminal carbonyl and the (HFIP)
C-H group is H-bonded to the penultimate carbonyl. Only in the
complex with molecule A is the -OH group of this same HFIP
molecule additionally H-bonded to the penultimate carbonyl, thus
generating two three-center H-bond motifs. In both complexes the
-OH group of each of the two sites of the second HFIP molecule
is H-bonded to the last but two carbonyl. Interestingly, this is the
carbonyl which would be involved in an intramolecular H-bond
with the C-terminal amide N-H group if the peptide would be
310- instead of R-helical. In all four HFIP molecules of the two
complexes the O-H and C-H bonds are syn periplanar, as reported
for the X-ray diffraction structure of the fluoroalcohol itself.13a There
are no H-bond interactions between HFIP molecules. Conversely,
the shortest F‚‚‚F separations between HFIP neighbors are 3.013-
(21) and 3.096(20) Å, respectively, in the two complexes. It has
been extensively demonstrated since 1964 that HFIP is a strong
H-bonding donor, thus being able to solvate and dissolve peptides,
proteins, and synthetic polyamides.13b-e H-bonding interaction
between cocrystallized HFIP and peptide molecules have been
previously reported only for two cyclic dimers.14 It is also worth
recalling that a single 2,2,2-trifluoroethanol (TFE) molecule coc-
rystallized with Ac-[L-(RMe)Val]8-OH is not able to displace this
homo-octapeptide free acid from the fully 310-helical conforma-
tion.15
Figure 1. X-ray diffraction structures of the three independent molecules
(A, B, and C) in the asymmetric unit of unsolvated Ac-[L-(RMe)Val]7-
NHiPr (1) (crystals grown from a MeOH solution). H-atoms have been
omitted for clarity. Dashed lines represent intramolecular CdO‚‚‚H-N
H-bonds.
1 and 7, and g+,t at position 2), different combinations of angles
(t,g-; g+,g-; g+,t) characterize the positions from 3 to 6. Overall,
the t,g- set prevails over the g+,t and g+,g- sets.7,12
A significant modification was observed in the 3D-structure of
the same peptide when the crystals were grown from an HFIP
solution, where it is R-helical (heptapeptide bis-HFIP solvate, 2).
The two independent peptide molecules (A and B) in the asym-
metric unit are almost identical, the corresponding φ,ψ backbone
torsion angles not differing more than 5° (Figure 2). Both are folded
in right-handed helical structures. The average φ,ψ torsion angles
for the seven residues are -54.9°,-50.8° (molecule A) and
-55.1°,-50.4° (molecule B). Interestingly, the ψ values are much
closer to those expected for an R-helix (-42°) than for a 310-helix
(-30°).1a Also the L-(RMe)Val side-chain dispositions are remark-
ably the same for molecules A and B, all sets of ø1,1 and ø1,2 torsion
angles being t,g- except those of residue 2 (g+,t).7,12 Finally, no
difference has been found in the intramolecular H-bonding
schemes: an i r i+3 hydrogen bond (indicative of a 310-helix) at
the N-terminus is followed by four, consecutive, i r i+4 hydrogen
bonds (typical of an R-helix). The range of O‚‚‚N separations is
2.941(11)-3.262(10) Å.11 The development of a fully formed
R-helical structure from an initial 310-helical nucleus is not
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C O M M U N I C A T I O N S
not involved in the CdO‚‚‚H-N intramolecular H-bonding net-
work. Both O-H‚‚‚O and C-H‚‚‚O types of H-bonds participate
in the solvation. The conformations of the peptide in the two crystals
strictly mirror those occurring in the two solvents. Interestingly,
Karle, Balaram, and their co-workers have already reported the
X-ray diffraction structures of the same, CR,R-di-n-propylglycine
containing, heptapeptide sequence (having a different N-terminal
group) in the 310- and R-helix conformations despite being
crystallized from the same solvent (the 310-helical structure is
monohydrated).16 The present investigation highlights that the
interconversion between R- and 310-helices might be allowed even
in peptides exclusively composed by CR-tetrasubstituted R-amino
acids and provides clues for a deeper understanding of the
interactions of HFIP with helical peptides.
Supporting Information Available: Preparative procedures and
characterization data; CD spectra; X-ray diffraction details, including
crystallographic data in CIF format. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 3. HFIP molecules bound to the carbonyl oxygen atoms in the
C-terminal region of the two independent peptide molecules (A and B) in
the asymmetric unit of the R-helical Ac-[L-(RMe)Val]7-NHiPr (2). Peptide
N-H and HFIP hydrogen atoms are shown (all other hydrogen atoms have
been omitted for clarity). Dashed lines represent intramolecular CdO‚‚‚
H-N hydrogen bonds, while dotted lines represent (HFIP) O-H‚‚‚OdC
(peptide) and (HFIP) C-H‚‚‚OdC (peptide) hydrogen bonds. Major and
minor occupancy sites for the hydroxyl group of one of the two HFIP
molecules bound to peptide A or B are indicated by solid and open C-O
bonds, respectively.
In conclusion, we have described an example of a solvent-driven
R/310-helix dimorphism for a peptide molecule in the crystalline
state. The fully CR-methylated homo-peptide Ac-[L-(RMe)Val]7-
NHiPr is completely 310-helical when its crystals are grown from
a MeOH solution. By contrast, it is folded in the R-helical
conformation when crystallized from HFIP, an alcohol of high
polarity. In this latter case, two cocrystallized solvent molecules
bind to the three C-terminal peptide (or amide) carbonyl functions
JA076656A
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