Stereoelectronic effects on collagen stability: The dichotomy of 4-fluoroproline diastereomers

Article abstract of DOI:10.1021/ja035881z

Collagen is the most abundant protein in animals. Natural collagen consists of a triple helix of (Xaa-Yaa-Gly)_n chains, in which the Xaa and Yaa residues are often l-proline. Here, a (2S,4S)-4-fluoroproline (flp) residue is shown to be greatly stabilizing in the Xaa position (but destabilizing in the Yaa position). In contrast, a (2S,4R)-4-fluoroproline (Flp) residue is shown to be greatly destabilizing in the Xaa position (but stabilizing in the Yaa position). The dichotomous effect of the diastereomers appears to arise from a gauche effect, which alters pyrrolidine ring pucker and hence properly (or improperly) preorganizes main-chain dihedral angles. Thus, the rational use of stereoelectronic effects can enhance the conformational stability of a protein. Copyright

Full text of DOI:10.1021/ja035881z

Published on Web 07/11/2003
Stereoelectronic Effects on Collagen Stability: The Dichotomy of
4-Fluoroproline Diastereomers
Jonathan A. Hodges† and Ronald T. Raines*,†,§
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received April 30, 2003; E-mail: raines@biochem.wisc.edu
Stereoelectronic effects can impose constraints on the conforma-
tion of organic molecules.1 Nonetheless, only one stereoelectronic
effect on the conformation of a protein is known.2 That unique
effect is manifested in collagen, the most abundant protein in
animals.
Collagen consists of three polypeptide chains that fold into a
triple helix.3 Each natural chain contains many repeats of the
sequence XaaYaaGly, in which a third of the Xaa and Yaa residues
are (2S)-proline (Pro) or (2S,4R)-4-hydroxyproline (Hyp).4 Replac-
ing Pro or Hyp in the Yaa position with the nonnatural amino acid
(2S,4R)-4-fluoroproline (Flp) greatly increases triple-helix stability.5
In contrast, replacing Pro or Hyp in the Yaa position with the
diastereomer (2S,4S)-4-fluoroproline (flp) greatly decreases stabil-
ity.2 Here, we use fluorine substitution to probe for stereoelectronic
effects in the Xaa position of collagen. Again, we find that one
diastereomer of 4-fluoroproline is greatly stabilizing to the triple
helix, and the other is greatly destabilizing. Remarkably, the
stereoelectronic preference of the Xaa position is opposite to that
of the Yaa position, as flp but not Flp endows hyperstability.
The pucker of a pyrrolidine ring can be influenced by electrone-
gative substituents.5,6 This effect is stereoelectronic, as it depends
on the configuration and electron-withdrawing ability of the
substituent.2,7 In particular, the gauche effect6b,8 exerted by an
electron-withdrawing 4R substituent stabilizes the Cγ-exo pucker,
and that by a 4S substituent stabilizes the Cγ-endo pucker. The
degree of stabilization is likely to be greatest for fluorine, the most
electronegative of atoms.
Molecular modeling of a triple helix of (ProProGly)10 strands
has suggested that Pro in the Xaa position prefers to adopt a Cγ-
endo pucker, whereas Pro in the Yaa position prefers a Cγ-exo
pucker.9 This pattern has been observed in a crystalline (ProPro-
Gly)10 triple helix.10 The pyrrolidine ring pucker influences the range
and distribution of the φ and ψ main-chain dihedral angles11 of
Pro and can fix those dihedral angles for optimal packing of the
triple helix. Increasing the preference for the desired Cγ-exo
conformation in the Yaa position by inclusion of either Hyp or Flp
decreases the entropic penalty for triple-helix formation. Likewise,
Hyp and Flp increase the preference of the ω main-chain dihedral
angle11 for the trans (ω ) 180°) conformation.2,6b,7a Because all of
the peptide bonds in collagen are trans, preorganization of ω by
Hyp and Flp decreases the entropic penalty for triple-helix
formation.
As in the Yaa position, preorganization of ω in the trans
conformation would also be favorable in the Xaa position. Yet, a
Cγ-exo conformation favors φ and ψ dihedrals that are not ideal
for this position. Hence, fixing the ring pucker of proline in the
Xaa position could have a favorable influence on either φ, ψ, or
ω, but not all three.
Figure 1. (A) Circular dichroism spectra at 5 °C. Solutions of peptide
(0.2 mM in 50 mM acetic acid) were allowed to incubate at 4 °C for g24
h before spectra were recorded. Inset: Structures of Flp and flp from ref 7.
(B) Thermal denaturation curves determined by measuring molar elipticity
at 225 nm as a function of temperature ((1 °C). Data were recorded at
intervals of 3 °C after equilibration for g5 min.
Can triple-helix stability stability be increased by fixing the ring
pucker of proline in the Xaa position? Replacing Pro in the Xaa
position of (ProProGly)10 with either Hyp12 or its diastereoisomer
(2S,4S)-4-hydroxyproline (hyp)13 is known to produce strands that
fail to form triple helices. This result could, however, be due to
unfavorable steric interactions that develop upon replacing a
hydrogen with a hydroxyl group. This suspicion is consistent with
molecular modeling of hyp in the Xaa position.9 Replacing hydrogen
with fluorine, on the other hand, typically results in little steric
destabilization.14
To search for a stereoelectronic effect in the Xaa position on
collagen stability, we again used fluorine as a probe, synthesizing
the peptides (FlpProGly)7 and (flpProGly)7. Circular dichroism
(CD)spectroscopy indicates that (flpProGly)7 but not (FlpProGly)7
forms a stable triple helix at 5 °C (Figure 1A). Moreover, only
(flpProGly)7 shows the cooperative transition characteristic of triple-
† Department of Chemistry.
§ Department of Biochemistry.
9
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J. AM. CHEM. SOC. 2003, 125, 9262-9263
10.1021/ja035881z CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Effects of 4-Hydroxyproline and 4-Fluoroproline
Diastereomers on the Conformational Stability of a Collagen Triple
Helix with (XaaYaaGly)7 Strands
the Xaa position. We anticipate that the rational use of stereoelec-
tronic effects could enhance the stability of other proteins as well.
Tm (°C)a
Acknowledgment. We are grateful to D. R McCaslin and C.
L. Jenkins for advice. 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 Wisconsin-Madison Biophys-
ics Instrumentation Facility, which was established by Grants BIR-
9512577 (NSF) and RR13790 (NIH).
Xaa/Yaa
(XaaProGly)7
(ProYaaGly)7
Flp
Hyp
Pro
hyp
flp
no helix
no helixc
6-7d
45b
36b
6-7d
no helixe
33
no helixe
no helixb
a Temperature at the midpoint of the thermal transition as measured by
CD spectroscopy. “No helix” refers to Tm < 5 °C. b From ref 2. c Reported
for (HypProGly)10 in ref 12. d From ref 15. e Reported for (hypProGly)10
and (ProhypGly)10 in ref 13.
Supporting Information Available: Procedures and additional data
for syntheses and analyses reported herein (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
helix stability unfolding upon thermal denaturation (Figure 1B).
The midpoint of this transition is at 33 °C (Table 1). The linear
decrease in elipticity exhibited by (FlpProGly)7 is characteristic of
the unfolding of a single polypeptide chain. Sedimentation equi-
librium experiments confirm that (FlpProGly)7 but not (flpProGly)7
is a single strand at 4 °C, whereas both peptides are single strands
at 37 °C.
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Apparently, stereoelectronic effects can operate adventitiously
(or deleteriously) in the Xaa position of collagen (Table 1). There,
flp is able to preorganize the φ and ψ dihedrals as in a triple helix
without encountering the steric conflicts that appear to plague hyp
in this position.9 Moreover, the 4S substituent in the Xaa position
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regions. Indeed, replacement of proline in the Xaa position with
Hyp does increase the stability of a proline-poor region.17
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