Origin of the stability conferred upon collagen by fluorination

Article abstract of DOI:10.1016/j.bmcl.2009.03.168

According to a prevailing theory, (2S,4R)-4-hydroxyproline (Hyp) residues stabilize the collagen triple helix via a stereoelectronic effect that preorganizes appropriate backbone torsion angles for triple-helix formation. This theory is consistent with th

Full text of DOI:10.1016/j.bmcl.2009.03.168

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3859–3862
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Origin of the stability conferred upon collagen by fluorination
Matthew D. Shoulders a, Kimberli J. Kamer b, Ronald T. Raines a,c,
*
a Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706-1322, USA
b Department of Biomedical Engineering, University of Wisconsin–Madison, 1550 Engineering Drive, Madison, WI 53706-1609, USA
c Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
According to a prevailing theory, (2S,4R)-4-hydroxyproline (Hyp) residues stabilize the collagen triple
helix via a stereoelectronic effect that preorganizes appropriate backbone torsion angles for triple-helix
formation. This theory is consistent with the marked stability that results from replacing the hydroxyl
group with the more electron-withdrawing fluoro group, as in (2S,4R)-4-fluoroproline (Flp). Nonetheless,
the hyperstability of triple helices containing Flp has been attributed by others to the hydrophobic effect
rather than a stereoelectronic effect. We tested this hypothesis by replacing Hyp with 4,4-difluoroproline
(Dfp) in collagen-related peptides. Dfp retains the hydrophobicity of Flp, but lacks the ability of Flp to pre-
organize backbone torsion angles. Unlike Flp, Dfp does not endow triple helices with elevated stability,
indicating that the hyperstability conferred by Flp is not due to the hydrophobic effect.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 12 March 2009
Accepted 30 March 2009
Available online 21 April 2009
Keywords:
Collagen
4,4-Difluoroproline
4-Fluoroproline
Hydrophobic effect
Peptide
Preorganization
Protein stability
Stereoelectronic effect
Triple helix
Collagen is the principal structural protein in animals. Collagen
is composed of a right-handed triple helix formed from three par-
allel polyproline type-II helices.1 The individual strands contain re-
peats of the sequence XaaYaaGly, where Xaa and Yaa are often
(2S)-proline (Pro) and (2S,4R)-4-hydroxyproline (Hyp), respec-
tively. The repetitive sequence, large size, and insolubility of native
collagen has motivated the study of triple-helix structure and sta-
bility with short (630-residue) collagen-related polypeptides
(CRPs). Much of our current understanding of the triple helix de-
rives from such studies.1
c
c
Figure 1. Ring conformations of Pro and its derivatives. The C -endo:C -exo ratio is
ꢀ2 when
R
1 = R2 = H (Pro).9 The C -exo conformation is favored strongly by
c
stereoelectronic effects when R1 = OH (Hyp) or F (Flp), R2 = H.6
Hyp is introduced in protocollagen strands by the post-transla-
tional hydroxylation of prolines in the Yaa position. This modifica-
tion, which provides dramatic conformational stability to triple-
helical CRPs,2,3 is essential for animal life.4,5 We have proposed that
Hyp stabilizes collagen via a stereoelectronic effect—the gauche ef-
are in accord with these conclusions. For example, CRPs containing
c
a diverse manifold of Pro derivatives with C -exo pucker in the Yaa
position are also hyperstable.12–15
A stereoelectronic origin for collagen stability has, however,
been questioned by others.16,17,34–36 In particular, Hyp and Flp have
been proposed to impart stability by distinct means. Differential
scanning calorimetry experiments show that the hyperstability of
triple helices formed from (ProFlpGly)10 is dominated by entropic
effects, whereas that of (ProHypGly)10 is dominated by enthalpic
effects.16,17 Because the hydrophobic effect is manifested in entro-
py, Flp has been proposed to stabilize the triple helix by the ener-
getically favorable segregation of hydrophobic fluorine atoms19
from water upon triple-helix folding. (It is noteworthy, however,
that triple-helix stabilization by preorganization would likewise
arise from an entropic effect.)
c
fect—which imposes a C -exo ring pucker6 upon the pyrrolidine
ring in Hyp and Flp residues (Fig. 1).7–10 Because the proline back-
c
bone torsion angles that accompany a C -exo ring pucker are re-
quired in the Yaa position of collagen triple helices, Hyp and Flp
could stabilize the collagen triple helix by preorganization of the
appropriate backbone dihedral angles for triple-helix forma-
tion.1,7,11 Flp is more effective than Hyp at this preorganization be-
cause fluorine is more electronegative than oxygen. Later findings
* Corresponding author. Tel.: +1 608 262 8588; fax: +1 608 262 3453.
E-mail address: raines@biochem.wisc.edu (R.T. Raines).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.168

3860
M. D. Shoulders et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3859–3862
The hydrophobic effect is a dominant force in the folding of
globular proteins.20 Moreover, substantial precedence exists for
enhancing protein stability by incorporating hydrophobic, fluori-
nated amino-acid residues within protein cores.21–25 The role for
the hydrophobic effect in collagen stability is, however, minimized
by collagen being a fibrous protein that lacks a substantive core. In-
deed, earlier studies suggested that the hydrophobic effect is not
important for triple-helix stability.26,27 Still, we sought to ascertain
whether triple-helix stabilization by Flp is due to a hydrophobic ef-
fect or a stereoelectronic effect. We reasoned that (2S)-4,4-difluo-
roproline (Dfp; Fig. 1 with R1 = R2 = F) would be useful in this
regard, because Dfp would exhibit a similar hydrophobic effect
but ambiguous stereoelectronic effects.
First, we determined whether Dfp and Pro do indeed have sim-
ilar preorganizational capacity. Substantial experimental evidence
supports this treatise. Moroder and co-workers used NMR spec-
Figure 2. Space-filling models of fluorinated triple helices constructed from the
three-dimensional structure of a (ProHypGly)n triple helix (PDB entry 1CAG18) by
replacing an OH, or OH and H of Hyp with F by using the program PyMOL (Delano
Scientific, Palo Alto, CA). (A) Segment of a model (ProFlpGly)n triple helix. (B)
Segment of a model (ProDfpGly)n triple helix.
c
troscopy to conclude that Ac-Dfp-OMe prefers the C -endo ring
pucker in water.28 Our own conformational analyses using 1H
and 19F NMR spectroscopy reveal a significant population of both
c
c
the C -exo and C -endo conformers of Ac-Dfp-OMe in both water
and chloroform (see Supplementary data), suggesting that the
c
c
C -exo and C -endo ring puckers of Dfp are of similar energy. This
finding indicates that the preorganizational capacity of Dfp is much
triple-helix stability are due primarily to the conformational pref-
erences of Pro derivatives, then replacing Pro with Dfp in a CRP
should have little effect on triple-helix stability. In contrast, replac-
ing Pro with Flp should enhance triple-helix stability. On the other
hand, if the partial burial of hydrophobic fluorine atoms is impor-
tant for the hyperstability of (ProFlpGly)n, then both Dfp- and Flp-
containing triple helices should have markedly enhanced thermal
stability relative to (ProProGly)n triple helices. Peptides appropri-
ate for this experiment were prepared by segment condensation
on a solid-phase using Fmoc-XaaYaaGly-OH amino acid trimers
prepared in solution (see Supplementary data). We could not ana-
lyze the conformational properties of the CRP (ProDfpGly)10 be-
cause of its poor solubility in water. Host–guest CRPs are
generally reliable models for the effects of amino acid substitution
on triple-helix structure and stability.27,30 Hence, we synthesized
the host–guest CRPs Ac-(ProProGly)10-NH2 (Pro-CRP), Ac-(ProPro-
Gly)4–ProDfpGly–(ProProGly)5-NH2 (Dfp-CRP), and Ac-(ProPro-
Gly)4–ProFlpGly–(ProProGly)5-NH2 (Flp-CRP). We introduced N-
terminal acetyl and C-terminal amido groups to eliminate unfavor-
able Coulombic interactions between the CRP termini.
c
closer to that of Pro (which has only a slight preference for the C -
c
endo ring pucker9) than to that of Flp (which prefers the C -exo
ring pucker strongly due to the gauche effect9).
Additional experimental evidence that Pro and Dfp have similar
triple-helix preorganizational capacity can be obtained by observ-
ing the equilibrium constant (Ktrans/cis) for the isomerization of
their peptide bonds. Typically, the Ktrans/cis value of a Pro derivative
is correlated to its ring pucker.9,28,29 Thus, Ktrans/cis values for Ac-
Xaa-OMe model systems can provide a valuable measure of the rel-
ative conformational preferences of Pro derivatives. We used 1H
NMR spectroscopy to show that Ac-Dfp-OMe has Ktrans/cis = 3.6 in
water (see Supplementary data). This value is similar to that of
Ac-Pro-OMe, which has Ktrans/cis = 4.6, but divergent from that of
Ac-Flp-OMe, which has Ktrans/cis = 6.7.7 Finally, replacement of Pro
with Dfp in a barstar variant does not alter its conformational sta-
bility, whereas the monofluorinated derivatives Flp and its diaste-
reomer, (2S,4S)-4-fluoroproline, have marked effects.28
Next, we resorted to hybrid density functional theory to explore
further the conformational preferences of Dfp. Geometry optimiza-
tions and frequency calculations were performed at the B3LYP/6-
311+G(2d,p) level of theory on four preferred conformations of
We analyzed the conformational stability of the triple helices
formed by the three CRPs with circular dichroism (CD) spectros-
copy. Values of Tm, which is the temperature at the midpoint of
the thermal transition, depend on CRP concentration.31 Accord-
ingly, the concentrations of stock solutions were determined by
amino-acid analysis and then adjusted to ensure that each CRP
was analyzed at the same concentration. After incubation at
64 °C for P24 h to allow triple helices to form, all three CRPs dis-
played the signature CD spectrum for a collagen triple helix at 4 °C,
with maxima near 225 nm (Fig. 3A). Upon heating, all three CRPs
underwent cooperative thermal transitions (Fig. 3B). We found
that the Dfp-CRP and the Pro-CRP triple helices had Tm values
within experimental error. The Tm value of a Flp-CRP triple helix
was, however, ꢀ6 °C higher (Fig. 3B). These data suggest that the
triple-helix stabilizing effects of Flp are attributable predominantly
c
Ac-Dfp-OMe in the gas phase. Briefly, we found that the C -endo
ring pucker is favored by 0.3 kcal/mol for Ac-Dfp-OMe in the cis
c
conformation, whereas the C -exo ring pucker is favored by
c
0.5 kcal/mol over a slightly distorted C -endo ring pucker in the
trans conformation. These calculated conformational preferences
are closer to those of Ac-Pro-OMe than to those of Ac-Flp-OMe.9
Hence, Pro and Dfp should have roughly similar triple-helix preor-
c
ganization capacity. Importantly, the C -exo conformations
adopted by Ac-Dfp-OMe have appropriate dihedral angles for the
Yaa position of the collagen triple helix, indicating that Dfp should
be acceptable in the Yaa position of a triple helix.
Then, we compared the effect of Dfp and Flp on triple-helix sta-
bility. Our model of a (ProFlpGly)n triple helix shows that the fluo-
rine atoms protrude tangentially from the triple helix and are
partially buried (Fig. 2A). In a hypothetical (ProDfpGly)n triple he-
lix, the fluorine atoms corresponding to those in a (ProFlpGly)n tri-
ple helix are buried to the same extent. The additional fluorine
atoms protrude radially from the triple helix and are exposed fully
to solvent (Fig. 2B). Because triple-helix folding partially buries one
fluorine atom per XaaYaaGly triplet in both CRPs, any stability aris-
ing from the hydrophobic effect should be similar in the two fluo-
rinated triple helices. Accordingly, if the effects of fluorination on
c
to its enhanced preference for the C -exo ring pucker. Apparently,
the thermodynamic advantage afforded by the partial burial of
hydrophobic fluorine atoms near the center of the triple helix is
minimal.32
Two caveats to this conclusion must be considered. First,
although fluorine is often regarded as an isosteric replacement
for hydrogen, it does have a larger covalent radius (rH = 0.31 Å;
rF = 0.57 Å).33 Hence, we must consider whether appending a sec-
c
ond fluoro group to C engenders any steric hindrance. Molecular

M. D. Shoulders et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3859–3862
3861
Award in Bioorganic and Medicinal Chemistry. The authors are
grateful to Dr. C. G. Fry for assistance with NMR experiments and
A. Choudhary for contributive discussions. This work was sup-
ported by Grant AR044276 (NIH). M.D.S. was supported by US
Department of Homeland Security and ACS Division of Medicinal
Chemistry graduate fellowships, and Chemistry–Biology Interface
Training Grant GM008505 (NIH). CD spectroscopy and mass spec-
trometry were performed at the University of Wisconsin–Madison
Biophysics Instrumentation Facility, which was established with
grants BIR-9512577 (NSF) and S10 RR13790 (NIH). NMR experi-
ments were performed at the National Magnetic Resonance Facility
at Madison, which is supported by Grant P41RR02301 (NIH), and
the University of Wisconsin Magnetic Resonance Facility, which
is supported by Grant CHE-9629688 (NSF).
A
5
0
Flp-CRP
Dfp-CRP Pro-CRP
–5
–10
–15
–20
–25
–30
220
230
240
250
260
210
wavelength (nm)
B
5
4
Supplementary data
Flp-CRP
3
Pro-CRP
Dfp-CRP
Supplementary data (detailed procedures for the syntheses and
analyses of Ac-Dfp-OMe and the peptides) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.03.168.
2
1
0
–1
–2
–3
–4
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Acknowledgments
This Letter is dedicated to Professor Carlos F. Barbas on the oc-
casion of his winning the 2009 Tetrahedron Young Investigator

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