CD and NMR investigation of collagen peptides mimicking a pathological Gly-Ser mutation and a natural interruption in a similar highly charged sequence context

Abstract

Even a single Gly substitution in the triple helix domain of collagen leads to pathological conditions while natural interruptions are suggested to play important functional roles. Two peptides-one mimicking a pathological Gly-Ser substitution (ERSEQ) and the other one modeling a similar natural interruption sequence (DRSER)-are designed to facilitate the comparison for elucidating the molecular basis of their different biological roles. CD and NMR investigation of peptide ERSEQ indicates a reduction of the thermal stability and disruption of hydrogen bonding at the Ser mutation site, providing a structural basis of the OI disease resulting from the Gly-Ser mutation in the highly charged RGE environment. Both CD and NMR real-time folding results indicate that peptide ERSEQ displays a comparatively slower folding rate than peptide DRSER, suggesting that the Gly-Ser mutation may lead to a larger interference in folding than the natural interruption in a similar RSE context. Our studies suggest that unlike the rigid GPO environment, the abundant R(K)GE(D) motif may provide a more flexible sequence environment that better accommodates mutations as well as interruptions, while the electrostatic interactions contribute to its stability. These results shed insight into the molecular features of the highly charged motif and may aid the design of collagen biomimetic peptides containing important biological sites.

Keywords: CD; NMR; collagen; interruption; mutation.

Figure 1

CD wavelength scans, thermal transitions and the first derivative (d(MRE)/dT) of the thermal transition curves of peptides (A–C) ERSEQ and (D–F) DRSER. Both peptides were prepared at a concentration of 1 mg/mL under two conditions: 20 mM PBS, 150 mM NaCl at PH 7 (gray), and 0.1M acetic acid, pH 3 (black). Wavelength scans were conducted at 4°C (A, D). Melting transitions were monitored by CD spectroscopy at 225 nm (B, E) and the first derivative of the thermal transition curves was calculated (C, F).

Figure 2

¹H–¹⁵N HSQC spectra of peptides ERSEQ (A) and DRSER (B) at pH 7 (red) and pH3 (green) at 25°C. Peptide sequences are shown at the top with ¹⁵N‐labeled residues underlined. The peaks corresponding to the monomer and trimer states are denoted with a superscript M or T, respectively. The superscripted numbers 1, 2, and 3 correspond to the three different chains, respectively.

Figure 3

³J_HNHα coupling values of model peptides ERSEQ (A) and DRSER (B). ³J_HNHα‐coupling measurements were performed at pH 7 (gray) and pH 3 (black) at 25°C. Residues in the triple helical conformation typically contain phi angles from −55 to −75° and have a corresponding J coupling value of 4–6 Hz.

Figure 4

Amide proton NH temperature gradients of peptide ERSEQ (A) and DRSER (B). Amide proton NH temperature gradients were measured at pH 7 (gray) and pH 3 (black). The black dashed horizontal line corresponds to a cut‐off value for hydrogen bonding, with less negative values than −4.6 ppb/°C indicative of hydrogen bonding.

Figure 5

CD and NMR folding curve of peptide ERSEQ (black) and DRSER (gray). The CD folding was monitored at pH 7 at a concentration of 1 mg/mL at 4°C (A). The residue‐specific NMR folding was monitored by measuring the intensity of Ser22 in the monomer state as a function of time at pH 7 (B).