C O M M U N I C A T I O N S
Table 1. Effect of 4-Methylproline and 4-Fluoroproline
Diastereomers on the Conformational Stability of the Collagen
Triple Helix
Security graduate fellowship. J.A.H. was supported by postdoctoral
fellowship AR48057 (NIH). This work was supported by Grant
AR44276 (NIH). CD and sedimentation equilibrium experiments
were performed at the University of WisconsinsMadison Biophys-
ics Instrumentation Facility, which was established by Grants BIR-
9512577 (NSF) and RR13790 (NIH).
peptide
Tm
(±
1
°
C)
ref
(Pro-Flp-Gly)7
(mep-Mep-Gly)7
(Pro-Hyp-Gly)7
(flp-Pro-Gly)7
(Pro-Mep-Gly)7
(mep-Pro-Gly)7
(flp-Flp-Gly)7
(Pro-Pro-Gly)7
45
36
36
33
29
13
3a
this work
3a
3c
Supporting Information Available: Procedures and additional data
for syntheses and analyses reported herein (PDF). This material is
this work
this work
17b
8a
-6a
17b
References
a Based on the extrapolation of data from solutions containing TMAO.
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(5) Analogous stereoelectronic effects have been observed in elastin-like
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P. J. Am. Chem. Soc. 2005, 127, 18121-18132.
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effect of the steric and stereoelectronic effects on the peptide bond
itself. The trans:cis ratio of Ac-Pro-OMe in D2O is only Ktrans/cis
) 4.6.3a Yet, all peptide bonds in the collagen triple helix are in
the trans conformation (ω ) 180°).6
To determine the effect of a 4-methyl group on the value of
K
trans/cis, we synthesized [13CH3]Ac-mep-OMe and [13CH3]Ac-
Mep-OMe and evaluated Ktrans/cis with 13C NMR spectroscopy. The
trans:cis ratio was 2-fold greater for Ac-Mep-OMe (Ktrans/cis
)
7.4) than for Ac-mep-OMe (Ktrans/cis ) 3.6). These data provide
an explanation for triple helices formed by (Pro-Mep-Gly)7 being
more stable than those formed by (mep-Pro-Gly)7.15 Apparently,
a balance exists between preorganization of the proper ring pucker
and stabilization of a trans peptide bond.16
Our findings have numerous implications. Only recently were
stereoelectronic effects found to contribute to the conformational
stability of a protein.3 Herein, steric effects are shown to reiterate
those same stereoelectronic effects. The stability of a non-natural
(mep-Mep-Gly)7 triple helix is indistinguishable from that of the
“natural” (Pro-Hyp-Gly)7 triple helix (Table 1), indicating that
side-chain heteroatoms (and hence side-chain solvation) are not
necessary for the formation of a stable triple helix. The stereoelec-
tronic effects induced by heteroatoms are not additive in collagen.
A (flp-Flp-Gly)7 triple helix is less stable than is a (flp-Pro-
Gly)7 or (Pro-Flp-Gly)7 triple helix (Table 1), presumably because
of an unfavorable steric interaction between fluoro groups on
adjacent strands.17 In contrast, the steric effects are additive, as a
(mep-Mep-Gly)7 triple helix is more stable than is a (mep-Pro-
Gly)7 or (Pro-Mep-Gly)7 triple helix (Table 1). The methyl groups
of mep and Mep in synthetic collagen can likely be elaborated to
larger functionalities without undesirable encumbrance. We imagine
the creation of a new class of hyperstable collagen mimetics by
the judicious integration of stereoelectronic and steric effects. The
application of these venerable principles coupled with recent
advances in the self-assembly of collagen fragments18 provides the
means to create sturdy synthetic collagens for applications in
biomedicine and biotechnology.
(9) Koskinen, A. M. P.; Helaja, J.; Kumpulainen, E. T. T.; Koivisto, J.;
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(10) It is noteworthy that a pseudoaxial Cγ-H allows for greater σ f σ*
hyperconjugative interactions with Cδ-N (a stereoelectronic effect) than
does a pseudoaxial Cγ-CH3. See: Weinhold, F.; Landis, C. R. Valency
and Bonding: A Natural Bond Orbital Donor-Acceptor PerspectiVe;
Cambridge University Press: Cambridge, UK, 2005.
(11) Del Valle, J. R.; Goodman, M. J. Org. Chem. 2003, 68, 3923-3931.
(12) See Supporting Information.
(13) CD experiments in solutions containing the osmolyte trimethylamine-N-
oxide confirm that triple helices of (mep-Pro-Gly)7 have a Tm value
near 13 °C, but the low molar ellipticity at 227 nm (Figure 2A) and the
results of sedimentation equilibrium experiments suggest that (mep-Pro-
Gly)7 is only partially assembled at 4 °C (ref 12).
(14) (a) Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Nature
1998, 392, 666-667. (b) Holmgren, S. K.; Bretscher, L. E.; Taylor, K.
M.; Raines, R. T. Chem. Biol. 1999, 6, 63-70.
(15) The value of Ktrans/cis for a 4-substituted proline residue correlates with its
ring pucker, an effect that is attributable to the stabilization of the trans
isomer by an n f π* interaction in the Cγ-exo conformation. See ref 3b
and Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188-1194.
(16) Mizuno, K. Hayashi, T.; Peyton, D. H.; Ba¨chinger, H. P. J. Biol. Chem.
2004, 279, 38072-38078.
(17) (a) Masamitsu, D.; Nishi, Y.; Uchiyama, S.; Nishiuchi, Y.; Nishio, H.;
Nakazawa, T.; Ohkubo, T.; Kobayashi, Y. J. Pept. Sci. 2005, 11, 609-
616. (b) Hodges, J. A.; Raines, R. T. J. Am. Chem. Soc. 2005, 127, 15923-
15932.
(18) (a) Paramonov, S. E.; Gauba, V.; Hartgerink, J. D. Macromolecules 2005,
38, 7555-7561. (b) Kishimoto, T.; Morihara, Y.; Osanai, M.; Ogata, S.;
Kamitakahara, M.; Ohtsuki, C.; Tanihara, M. Biopolymers 2005, 79, 163-
172. (c) Koide, T.; Homma, D. L.; Asada, S.; Kitagawa, K. Bioorg. Med.
Chem. Lett. 2005, 15, 5230-5233. (d) Kotch, F. W.; Raines, R. T. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 3028-3033.
Acknowledgment. We are grateful to F. W. Kotch, J.-C. Horng,
D. R. McCaslin, F. Weinhold, and M. C. Pirrung for helpful
discussion. M.D.S. was supported by a Department of Homeland
JA061793D
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J. AM. CHEM. SOC. VOL. 128, NO. 25, 2006 8113