Site‐Directed Spin Labeling of a Collagen Mimetic Peptide

Abstract

Collagen as the most abundant protein in mammals consists of three chains that are built up of repeating Xaa-YaaGly units with all peptide bonds in trans conformation and most often with proline in Xaa and (4R)-hydroxyproline in the Yaa position. This type of sequence favors a lefthanded poly-Pro-II helix conformation and the intertwining of the three chains into the unique right-handed triple helix. The X-ray structure analysis of collagen mimetic peptides (CMPs) of repeating Xaa-Yaa-Gly triplets show Cendo puckers of the Pro residues in Xaa and C-exo puckers of the Hyp residues in Yaa position. Studies on structural factors that stabilize the triple helical fold with proline derivatives led to the conclusion that besides the interchain hydrogen bond between the NH group of Gly of one strand and the CO group of Pro of the adjacent strand, stereoelectronic effects that favor the all trans-peptide bond conformation and the correct puckering are decisively affecting the structural stability. Recent studies by Erdman and Wennemers with proline derivatives in the Yaa position that exhibit preferences for the C-endo puckering, but a trans amide conformer, contradict this conclusion showing that the ring puckering is less important for the stability of collagen if the trans/cis amide conformer ratio favors formation of the triple helix. The aim of the present study was to investigate whether electron spin resonance spectroscopy (ESR) could yield new information on the dynamics of triple helix folding/unfolding as a useful alternative to the other spectroscopic techniques applied so far. Indeed this type of spectroscopy has become popular for studying conformational changes and unfolding processes of proteins as many ESR spectral parameters, such as peak-to-peak height, peak-to-peak width and rotational correlation time can accurately reflect relative mobility and environmental changes of spin labels (Figure 1). For the design and synthesis of a suitable spin-labeled CMP, the results of a recent detailed study on the equilibrium constants of acetyl-(4R)-Pro(X)-OMe (X=ammonium or acetylamide) by NMR spectroscopy were taken into account. Although for the ammonium derivative a significantly reduced preference for the trans conformation was observed, a trans conformer preference similar to that of (4R)hydroxyproline was recovered upon acetylation of the gamino group. Correspondingly, Ac-(Gly-Pro-Hyp)7-Gly-GlyNH2 [7] was selected as the CMP in which the Hyp residue of the central triplet was replaced by (2S,4R)-aminoproline [a] J. Jiang, Q. Jin, W. Ma, Prof. Dr. S. Dong School of Life Sciences, Lanzhou University 222 Tianshui South Road, Lanzhou 730000 (China) E-mail : dongsl@lzu.edu.cn [b] L. Yang Department of Medical Laboratory and Research Center Tangdu Hospital, Fourth Military Medical University Xi’an 710038 (China) [c] Prof. Dr. L. Moroder Bioorganic Chemistry, Max Planck Institute of Biochemistry Am Klopferspitz 18, 82152 Martinsried (Germany) [d] Prof. Dr. S. Dong Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, 222 Tianshui South Road Lanzhou 730000 (China) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303290. Figure 1. The relative mobility and environmental changes of spin labels with collagen folded/unfolded states.