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

Conformational Stability of Collagen Relies on a Stereoelectronic Effect

(1980). Intramolecularly Hydrogen-Bonded Peptide Conformation. Critical Reviews in Biochemistry: Vol. 9, No. 1, pp. 1-44.

Intramolecularly hydrogen-bonded peptide conformations.

For conjugates Ac-Hel1-Alan-OH, n = 1−6, of the previously characterized reporting, conformational template Ac-Hel1, increases in helicity induced by trifluoroethanol (TFE) in water have been related to a simple function of the peptide length n, yielding the helix propagation constant sAla, which increases from 1.0 to 1.5 for χTFE = 0−20 mol %. The per-residue helicity increase is similar to the increase in te state stability induced by TFE in monoamide conjugates Ac-Hel1-NHR. Addition of TFE to water significantly increases the rate of interconversion of s-cis/s-trans amide conformers for Ac-Pro-NHMe, consistent with a significant and selective destabilization of the planar resonance-stabilized amide. In dilute aqueous solution TFE increases helicity by selectively destabilizing amide functions that are solvent exposed, with the consequence that compact conformations such as helices that maximize intramolecular amide−amide hydrogen bonding and minimize amide solvent exposure are selectively favored.

Mechanism of Stabilization of Helical Conformations of Polypeptides by Water Containing Trifluoroethanol

The well-known preference of the peptide bond for the trans conformation has been attributed to steric effects. Here, we show that a proline residue with an N-formyl group (Hi−1−C′i−1=Oi−1), in which Hi−1 presents less steric hindrance than does Oi−1, likewise prefers a trans conformation. Thus, the preference of the peptide bond for the trans conformation cannot be explained by steric effects alone. Rather, an n → π* interaction between the oxygen of the peptide bond (Oi−1), and the subsequent carbonyl carbon in the polypeptide chain (C′i) also contributes to this preference. The Oi−1 and C′i distance and Oi−1···C′i=Oi angle are especially favorable for such an n → π* interaction in a polyproline II helix. We propose that this electronic effect provides substantial stabilization to this and other elements of protein structure.

https://onlinelibrary.wiley.com/doi/pdf/10.1110/ps.0241903

An electronic effect on protein structure.

The hydroxylation of proline residues in collagen increases the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res. 1994 , 44, 262−269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[β,γ-13C]d,l-proline methyl ester (1); N-acetyl-4(R)-hydroxy-l-proline [13C]methyl ester (2); and N-acetyl-4(R)-fluoro-l-proline methyl ester (3). The pKa's of the nitrogen atom in the parent amino acids decrease in the following order:  proline (10.8) > 4(R)-hydroxy-l-proline (9.68) > 4(R)-fluoro-l-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the following order:  1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater...

/pdf/inductive-effects-on-the-energetics-of-prolyl-peptide-bond-4j6ch25jiu.pdf

Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability.

The crystal structure of acetyl-L-proline-N-methylamide has been determined by the symbolic addition method. The crystal is orthorhombic, space group P21212x with four molecules in a cell of dimensions a = 9.74_+ 0.01, b = 13.20 +__ 0.01, c = 7.17 _+ 0.01/~. Refinement was carried out by the full-matrix leastsquares method including anisotropic thermal parameters. The final R value for 1213 reflexions was 0.098. The main chain of the molecule containing two trans planar peptide groups takes a folded conformation, the dihedral angle between the two peptide planes being 69.4 °. The two imino nitrogen atoms are arranged nearly in cis position and the torsion angles, tp and g are 103.7 and 164.2 ° respectively, indicating that the conformation is fairly close to that of the right-handed e-helix. The molecules are bound together through N H . . . O hydrogen bonds to form a chain about the twofold screw axis parallel to c.

T. Matsuzaki

Papers

The crystal structure of acetyl-l-proline-N-methylamide