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

The hydroxylation of proline residues in collagen is the most common posttranslational modification in humans. The hydroxylation 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 triplehelix 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)-4chloroproline.5 This explanation has been challenged by a hostguest 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 comparison. Then, we synthesized (2S,4R)-4-methoxyproline (Mop)15 and incorporated it into a collagen-related peptide: (ProMopGly)10 (2). We then used circular dichroism (CD) spectroscopy to discern the 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 Next, we used differential scanning calorimetry (DSC) to reveal the thermodynamic basis for the greater conformational stability 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 thermodynamic 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. 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 † Department of Chemistry. ‡ Present address: Chaperone Technologies, Inc., 66 Analomink Street, East Stroudsburg, PA 18301. § Department of Biochemistry. Figure 1. CD spectroscopy and DSC data for peptides 1 and 2. (A) CD spectra of 1 (O) and 2 (b) (100 μM) at 4 °C in 50 mM HOAc (pH 3.0). (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. Published on Web 02/14/2008