Conformational stability of collagen relies on a stereoelectronic effect.

Abstract

A polypeptide chain can adopt many conformations. Yet, the sequence of its amino acid residues directs folding to a particular native state.1 The loss of conformational entropy associated with folding destabilizes the native state. This destabilization is overcome by the hydrophobic effect, hydrogen bonds, other noncovalent interactions, and disulfide bonds.2 We have identified another force that can contribute to the conformational stability of a protein. The structure and reactivity of an organic molecule can rely on the stereochemistry of its electron pairs, bonded or nonbonded.3 Such stereoelectronic effects, which arise from the mixing of an electron pair with the antibonding σ* of an adjacent polar bond (C-X, where X ) N or O), endow nucleic acids and carbohydrates with conformational stability.4 For example, the multiple gauche effects (X-C-C-X) arising from a 2′ oxygen distinguish RNA‚RNA and RNA‚DNA duplexes from DNA‚DNA duplexes.5 The anomeric effect (X-C-X) enhances the stability of the R anomer of glycosides.6 Here, we demonstrate for the first time that a stereoelectronic effect is critical for the conformational stability of a protein. Collagen is the most abundant protein in animals.7 For example, collagen comprises one-third of the protein in humans and threefourths of the weight of human skin. The polypeptide chains of collagen are composed of approximately 300 repeats of the sequence XaaYaaGly, where Xaa is often an L-proline (Pro) residue and Yaa is often a 4(R)-hydroxy-L-proline (Hyp) residue. These chains are wound in tight triple helices, which are organized into fibrils of great tensile strength. Pro and Hyp comprise nearly one-fourth of the residues in common types of collagen.8 This prevalence of tertiary amides has dichotomous consequences for conformational stability. Pro and Hyp residues are constrained by their pyrrolidine rings, and this rigidity stabilizes triple-helical collagen.9 Yet, the trans and cis conformations of the peptide bonds to Pro and Hyp residues are of nearly equal free energy, which destabilizes collagen because all peptide bonds in triple-helical collagen are in the trans conformation (Figure 1).10 The 4(R)-hydroxyl group of the prevalent Hyp residues increases dramatically the conformational stability of collagen.11 We had shown previously that this increase arises from inductive effects.12 Can a stereoelectronic effect influence Ktrans/cis? To answer this question, we synthesized residue mimics of the form AcYaaOMe, where Yaa is Pro, Hyp, 4(S)-hydroxy-L-proline (hyp), 4(R)-fluoroL-proline (Flp), or 4(S)-fluoro-L-proline (flp).13 We chose Flp and flp because fluorine is small and electronegative and forms only weak hydrogen bonds when bound to carbon.14,15 We chose a methyl ester rather than an amide to prevent γ-turn formation, as had been observed in AcProNHMe.16 We find that the electronegativity and stereochemistry of the 4-substituent in the Yaa mimics has a significant effect on Ktrans/cis (Table 1). Compared to a flp residue, a Pro residue is twice as likely and a Flp residue is 3 times as likely to have a trans peptide bond. Does the value of Ktrans/cis have an impact on collagen stability? To answer this question, we synthesized (ProYaaGly)7 strands containing Flp or flp residues in the Yaa position.19 These strands are diastereomeric, differing only in the stereochemistry at Cγ of the Yaa residues. We found that a (ProFlpGly)7 triple helix has a Tm of 45 °C.20 In contrast, a (ProflpGly)7 triple helix has a Tm of <2 °C (Table 1). A (ProHypGly)7 triple helix has an intermediate Tm of 36 °C. Thus, both the electronegativity and