Protein flexibility is intimately related to protein function. A thorough understanding of the relationship between protein flexibility, structure, and amino-acid sequence would improve one's ability to predict a proteins function from knowledge of its amino-acid sequence alone. The goal of this project is to decipher the role of protein flexibility in collagenolysis. X-ray crystallographic structures of collagen-like model peptides suggest that collagens triple-helical structure cannot fit into the collagenase binding site. Moreover, these data imply that the scissile bond which forms the collagenase cleavage site is hidden from solvent and therefore inaccessible to collagenases. Therefore, the specific hypothesis that forms the basis of this work is that the precise amino acid sequence near the unique collagenase cleavage site imparts significant conformational flexibility to the structure of collagen. This structural ability enables sequences near the collagenase cleavage site to adopt conformations where the scissile bond is exposed and amenable to cleavage. Hence this project is designed to address a fundamental problem in biology; i.e., how do collagenases recognize their cleavage sites in collagen. While x ray crystallography and nuclear magnetic resonance (NMR) experiments have provided valuable insights into the structure of collagen and collagen-like model peptides, these methods may not fully capture the thermodynamic properties of collagen at physiologic temperatures. Prior studies on collagen-like model peptides, for example, were performed at temperatures at or below 10c. Such data provide insights into the structure of collagen at temperatures where the native triple-helical structure is most stable. However, the melting point of tropocollagen (the molecular unit that makes up collagen fibers) in vitro is slightly below body temperature, suggesting that micro-unfolding may play a role in the normal functioning of collagen fibers. Given these observations, it is likely that studies designed to probe the thermodynamics of collagen-like model peptides at temperatures near their melting temperature provide more relevant information about the structure of collagen at body temperature. This project combines molecular simulations with focused biochemical and NMR experiments to construct detailed models of the structure of collagen at different temperatures. Molecular simulations have the advantage of providing a window into the precise interactions underlying biochemical phenomena at an atomistic level of detail. Nevertheless, as the accuracy of these results depends on the accuracy of the underlying potential function, these data need to be verified experimentally. Consequently, the aims of this project are to: (1) compute conformational free energy profiles at different temperatures for a series of peptides that model regions of collagen near potential collagenase cleavage sites; (2) use the free energy profiles to calculate hydrogen deuterium-exchange protection factors (HDPFs) for amide nitrogens and secondary chemical shifts (SCSs) of Ca carbons in collagen-like model peptides; (3) validate and improve the results of the molecular simulations with experimentally determined HDPFs and SCS; and (4) use the resulting free-energy profiles to deduce sequence features that enable collagenases to recognize and cleave collagen.
Broader impacts of the project are to bridge the gap between research and teaching using a framework where students actively participate in scientific investigation. This project is designed such that there is a role for high school students, undergraduates, and graduate students. Minority high-school students in the Boston area will be actively recruited each summer to work on aspects of this research. These students will conduct simple biochemical experiments to characterize each of the collagen-like peptides under study and will also take an introductory course on protein structure, designed specifically for them. The goal is to establish a long term mentor-mentee relationship with these students that will persist throughout their academic life.
During the award period we have conducted a number of studies that have clarified the relationship between thermal fluctuations in collagen and collagenolysis. In particular: 1) Our work suggests that collagen can adopt partially unfolded states in solution and that this partial unfolding explains many aspects of collagenolysis that were previously mysterious. To accomplish this we used a combination of detailed molecular simulations coupled with biochemical experiments. 2) We have shown that collagen-like peptides can modulate the innate immune system in human monocytes. To our knowledge, this was the first study to do so. 3) Several papers have been produced as part of this work: T. Gurry, P. S. Nerenberg and C. M. Stultz, Biophys J 98 (11), 2634-2643 (2010). P. S. Nerenberg, R. Salsas-Escat and C. M. Stultz, Proteins 70 (4), 1154-1161 (2008). P. S. Nerenberg and C. M. Stultz, J Mol Biol 382 (1), 246-256 (2008). R. Salsas-Escat and C. M. Stultz, Exp Mech 49 (1), 65-77 (2009). R. Salsas-Escat, P. S. Nerenberg and C. M. Stultz, Biochemistry-Us 49 (19), 4147-4158 (2010). R. Salsas-Escat and C. M. Stultz, Proteins 78 (2), 325-335 (2010). Lu KG., Stultz CM., Insight into the Degradation of Type-I Collagen Fibrils by MMP-8. Journal of Molecular Biology (in press, published online ahead of print: http://dx.doi.org/10.1016/j.jmb.2013.02.002), 2013. 3) Two PhD students were trained during these activities in addition to 5 undergraduate students. One of these PhD students is now a tenure track faculty member at Claremont McKenna College.
