Stabilization of the collagen triple helix by O-methylation of hydroxyproline residues

Article abstract of DOI:10.1021/ja800225k

Collagen is the most abundant protein in animals, including humans. The prevalent (2S,4R)-4-hydroxyproline (Hyp) residues of collagen are known to confer great stability upon its triple-helical conformation. The basis for that stability has been attribute

Full text of DOI:10.1021/ja800225k

Published on Web 02/14/2008
Stabilization of the Collagen Triple Helix by O-Methylation of Hydroxyproline
Residues
Frank W. Kotch,†,‡ Ilia A. Guzei,† and Ronald T. Raines*,†,§
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received January 14, 2008; E-mail: rtraines@wisc.edu
The hydroxylation of proline residues in collagen is the most
common posttranslational modification in humans. The hydroxy-
lation is stereoselective, affording (2S,4R)-4-hydroxyproline (Hyp)
in the Yaa position of the canonical Xaa-Yaa-Gly triad and
thereby bestowing marked stabilization upon the collagen triple
helix.1 The means by which Hyp stabilizes collagen has engendered
dispute. One hypothesis suggests that a network of water molecules
links the Hyp hydroxyl groups and main-chain carbonyl groups.2,3
An alternative hypothesis invokes a stereoelectronic effect by which
the electronegative oxygen preorganizes the main chain in the proper
conformation for triple-helix formation.4
The latter explanation originates from the observation that
replacing Hyp with (2S,4R)-4-fluoroproline (Flp) increases triple-
helix stability; the fluoro group is strongly electron-withdrawing
but cannot participate effectively in a putative hydrogen-bonded
network. Similar results have been obtained with (2S,4R)-4-
chloroproline.5 This explanation has been challenged by a host-
guest study in which a single Hyp f Flp substitution was shown
to destabilize a triple helix.6 A similar study has, however, reported
a stabilization.7 So the question remains: does Hyp stabilize
collagen by serving as a template for a water network or through
stereoelectronic effects?
To differentiate between these hypotheses, we have made perhaps
the simplest of covalent modifications to Hyp: O-methylation.
Similar alkylations are known to decrease the hydration of
alcohols,8,9 nucleobases,10 and phospholipids.11 Yet, O-methylation
conserves the stereoelectronic effects of a hydroxyl group, as the
electron-withdrawing12 and hyperconjugative ability13 of OH and
OCH3 are similar. Moreover, the O-methylation of Hyp introduces
less steric encumbrance than does O-acetylation, which is known
to destabilize the collagen triple helix.14
We used commercial (ProHypGly)10 (1) as a basis for compari-
son. Then, we synthesized (2S,4R)-4-methoxyproline (Mop)15 and
incorporated it into a collagen-related peptide: (ProMopGly)10 (2).
Figure 1. CD spectroscopy and DSC data for peptides 1 and 2. (A) CD
We then used circular dichroism (CD) spectroscopy to discern the
spectra of 1 (O) and 2 (b) (100 µM) at 4 °C in 50 mM HOAc (pH 3.0).
effect of O-methylation. Peptides 1 and 2 were observed to form a
triple helix at 4 °C, as evidenced by a weak positive CD signal
near 225 nm and a strong negative signal near 200 nm (Figure
1A). In addition, both were found to undergo cooperative transitions
upon heating (Figure 1B), indicative of an unfolding triple helix.
Most interestingly, triple helices of 2 were discovered to have
substantially more conformational stability than those of 1 (Table
1). As in water, 23 was found to be more stable than 13 in aqueous
ethylene glycol (EG; Figure 1B, Table 1), which is known to
stabilize the collagen triple helix.4c,16
(B) Thermal denaturation of 1 and 2 (200 µM) in 50 mM HOAc(aq) (O,
b) and 2:1 EG/50 mM HOAc (pH 3.0) (], [). (C) DSC scans of 1 (231
µM) and 2 (129 µM) in 50 mM HOAc (pH 3.0); scan rate ) 15 °C/h.
of triple-helical 2. The stability of 13 relies more on enthalpy and
less on entropy than does that of triple-helical (ProFlpGly)10 (3),
indicative of a lesser reliance on a water network.17 The thermo-
dynamic parameters for 23 lie between those for 13 and 33 (Figure
1C; Table 1), suggesting that 23 is hydrated to an intermediate
extent. The decrease in hydration and increase in conformational
stability in the series 13 f 23 f 33 is consistent with hydration
being deleterious, rather than advantageous, to the collagen triple
helix.
Next, we used differential scanning calorimetry (DSC) to reveal
the thermodynamic basis for the greater conformational stability
† Department of Chemistry.
Finally, we determined the effect of the methoxy group on the
conformation of a Mop residue. To do so, we synthesized the model
compound Ac-Mop-OMe and determined its crystal structure
‡ Present address: Chaperone Technologies, Inc., 66 Analomink Street, East
Stroudsburg, PA 18301.
§ Department of Biochemistry.
9
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J. AM. CHEM. SOC. 2008, 130, 2952-2953
10.1021/ja800225k CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Thermodynamic Data for the Unfolding of Collagen
Triple Helices
are the other two amino acids containing a hydroxyl group, Ser
and Thr,21 and host-guest studies indicate that Ser and Thr are
not especially beneficial to collagen stability.22 Thus, we believe
that O-methylation could be a simple means to stabilize natural
collagen and, thereby, enhance its utility as a biomaterial.23
circular
dichroism
DSC
T
m, watera
C)
T
m, EG(aq)b
C)
∆
H
T
∆
S
∆G
peptide
sequence
(
°
(
°
(kcal/mol) (kcal/mol) (kcal/mol)
Acknowledgment. We are grateful to M. D. Shoulders and D.
R. McCaslin for contributive discussions. This work was supported
by Grant AR044276 (NIH) and Instrumentation Grants BIR-
9512577 (NSF) and RR13790 (NIH). F.W.K. was supported by
Postdoctoral Fellowship AR050881 (NIH).
1
2
3
(ProHypGly)10
(ProMopGly)10
(ProFlpGly)10
62.0
70.1
91d
77.1
89.1
ND
-35.2c -33.2c -2.0c
-27.9 -25.2
-2.7
-20.5c -17.2c -3.3c
a
b
c
50 mM HOAc (pH 3.0). 2:1 EG/50 mM HOAc (pH 3.0). Values
d
from ref 17. Value from ref 4a. ND ) Not determined.
Supporting Information Available: Procedures and additional data
for syntheses and analyses reported herein. Full citation for ref 15. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. (A) Molecular drawing of crystalline Ac-Mop-OMe (50%
probability ellipsoids). (B) Conformation of crystalline Ac-Mop-OMe and
Ac-Hyp-OMe showing the putative n f π* interaction.
Table 2. Values of φ, ψ, ω, and Kt/c for Ac-Mop-OMe and
Analogues
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b
parameter
Ac-Mop-OMe
Ac-Hyp-OMea
Ac-Flp-OMea
13
φ (deg)
ψ (deg)
ω (deg)
Kt/c
-58.1 ( 0.1
147.7 ( 0.1
-179.7 ( 0.1
6.7 ( 0.3c
-57.0
150.8
-178.8
6.1
-55.0
140.5
-178.9
6.7
-59.6
149.8
178.5
∞
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a
Mean values of φ, ψ, and ω from two molecules in ref 18; values of
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are σI ) 0.26 for OH and 0.22 for OCH3. See: Janesko, B. G.; Gallek, C.
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K
CD3OD by 13C NMR spectroscopy using [13CH3]Ac-Mop-OMe.
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(Figure 2A). The pyrrolidine ring of Mop adopts a Cγ-exo ring
pucker, which likely derives from a gauche effect between Ni and
Oδ1 4c,18,19 In addition, the conformation of Ac-Mop-OMe appears
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.
i
to rely on another stereoelectronic effect; the Oi-1‚‚‚C′idOi distance
of 2.84 Å and Oi-1‚‚‚C′idOi angle of 94.6° are indicative of a
favorable n f π* interaction (Figure 2B).4c,20 This stereoelectronic
effect would stabilize the trans (Z) isomer of the amide bond in
Ac-Mop-OMe. Indeed, Ac-Mop-OMe has a trans/cis ratio of Kt/c
) 6.7 (Table 2), which is among the largest reported in a derivative
of Ac-Pro-OMe.1 Thus, these two stereoelectronic effects appear
to preorganize the main-chain dihedral angles of Ac-Mop-OMe (as
well as Ac-Hyp-OMe and Ac-Flp-OMe) close to those in 13 (Table
2).
The conformational stability conferred upon the collagen triple
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group of Hyp acts primarily through stereoelectronic effects and
that its hydration provides little (if any) benefit. This finding could
have practical consequences. Replacing a hydroxyl group in a
protein with a fluoro group while retaining the stereochemical
configuration (as in Hyp f Flp) is not possible with extant reagents.
In contrast, O-methylation is a readily achievable transformation.
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