Collin M. Stultz - US grants

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.

Through a grant from the National Science Foundation to the Massachusetts Institute of Technology, six faculty members will collaborate in the purchase and use of high-performance computing equipment for research and education in computational and systems biology. The advent of high-throughput technologies in the life sciences has provided many genome sequences, protein structures and biological interaction networks. The amount of such data will continue to grow, compelling the development of rigorous and quantitative approaches to decipher and understand it. Simultaneously, advances in computing technology are enabling new ways of attacking complex biological problems using modeling and simulation. The projects to be supported cover a wide range of exciting areas, including the study of gene and organism evolution, transcriptional and post-transcriptional gene regulation, molecular signaling, protein conformational modeling, protein design, and the analysis of complex networks. The work will lead to advances in computational methods and provide basic biological insights.

This award will support MIT?s active role in developing computational and systems biology in the United States. The university is establishing novel programs and curricula to train students at the interface of the life sciences, engineering and the physical sciences. The investigators on this award are deeply involved in these activities. Shared computing resources will help attract talented students and provide them with modern, cross-disciplinary training. These students, who come from diverse backgrounds, will assume leadership positions in American universities and companies.

DESCRIPTION (provided by applicant): Tau is a natively unfolded protein that has been implicated in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease (AD). A number of studies suggest that aggregates of tau contribute to neuronal death and dysfunction in AD patients. Therefore, obtaining information about structural features that enable tau to form aggregates is of immense importance. In this proposal we develop and test novel approaches for constructing ensembles that adequately represent the unfolded state of tau. Our objective is to establish a new paradigm for building ensembles that adequately model the accessible conformations of intrinsically disordered proteins like tau. Using these methods we identify structural features within the unfolded ensemble of tau that are associated with aggregation. The goals of this proposal are to: 1) Generate structural ensembles that adequately represent accessible conformations of tau using computational methods (e.g., molecular dynamics simulations). Experimental data, such as chemical shifts, are used to guide the generation of appropriate ensembles;2) Generate similar structural ensembles for tau mutants, and phosphorylated forms of tau, that are associated with increased aggregation. Potential aggregation-prone conformers are identified by comparing ensembles corresponding to both native and aberrant proteins. Once aggregation-prone conformers have been identified, we will estimate the relative ability of potential aggregation-prone conformers to self-associate. Our long term goal is to use the information obtained from these studies to design small molecules that prevent the self-association of aggregation-prone conformers. PUBLIC HEALTH RELEVANCE: A number of studies suggest that aggregates of tau protein contribute to neuronal death and dysfunction in patients with Alzheimer's disease. We propose to identify structural features that enable tau to form aggregates in solution. Our long term goal is to use this insight to design potential therapies that prevent the formation of tau aggregates.

DESCRIPTION (provided by applicant): This is an application for partial support of the Collagen Gordon Research Conference, and the associated Gordon Research Seminar (GRS), in 2013. It will be the 41st anniversary and the 26th meeting of this premier conference and the second meeting of the Collagen GRS. It will be held at Colby-Sawyer College in New London, NH, July 13th to 19th. The conference has historically provided a unique forum to bring together an international group of junior and senior scientists with a common interest in the chemistry, biology and pathology of connective tissues, and this upcoming conference is no exception. Collaborations are established, new scientists are recruited to the field, and major discoveries about collagen are often reported for the first time at this conference. The specific aims of the 2013 collagen GRC are: 1) to communicate and disseminate new data and concepts about the chemistry, biology and pathology of collagens; 2) to promote interactions and collaborations among basic research groups, clinical research groups, and pharmaceutical and biotechnology companies; 3) to familiarize investigators with advances in biological concepts and emerging technologies in different fields that may be applied to the collagen field; 4) to provide and informal atmosphere for discussion of new hypotheses and approaches, and use of new enabling technologies; 5) to provide a collegial and highly interactive atmosphere in which both junior and senior investigators may interact; and 6) to increase the number and diversity of students and fellows attending the conference. The title of the 2013 Conference will be Collagen: In the context of matrix, cells and regenerative medicine. We will focus on new unpublished findings on collagens with respect to their role in both normal and disease processes. Importantly, new session topics have been integrated into the traditional program to bolster interest, increase applicants for the conference, ensure enthusiasm throughout the week, and promote major advancements in the field. Additionally, each session will feature graduate students/fellows selected from the Gordon Research Seminar (GRS) that is to be held in conjunction with the 2013 Collagen GRC. New topics relative to last year's conference include Genetic Mechanisms of Disease, Collagen Regulation and Dysregulation, and Future Directions in collagen matrix research. This latter section will showcase new and provocative work at the forefront of the field. We have also included updates on important topics such as Stem Cells, Repair and Regenerative Medicine, Matrix Mechanobiology and Collagen-Protein Interactions and Signaling. Overall this international conference will showcase the major advances in the field of collagen research and will be, as it has always been, the seminal conference in the field.