Structure, Dynamics and Energetics of Protein Unfolded States

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The native state of a protein is the structure that is most thermodynamically stable and is normally the biologically active form. Protein folding begins from a less structured collection of states, commonly known as the denatured state ensemble (DSE). The DSE is considerably harder to study than the native state owing to its dynamic and flexible nature, and to the fact that the vast majority of protein molecules populate the native state under normal conditions. Nevertheless, understanding the DSE is fundamentally important for understanding of protein folding and stability. The stability of a protein is dictated by the free energy difference between the native state and the DSE, thus any changes in solution conditions or mutations that alter the DSE can alter stability and folding. The DSE is the reference state for stability measurements; much of our quantitative understanding of the specific interactions involved in stabilizing the native state of proteins is derived from experiments that make implicit assumptions about the DSE. In addition, the DSE can be the starting state for nonproductive protein aggregation, a process that impacts biotechnology and biology. This project is directed at fundamental studies of protein folding, focusing on the role of the DSE. A deeper understanding of the properties of the DSE and the role that the DSE plays in folding is required for a complete description of protein folding and aggregation. The DSE can be populated under harsh, non-physiological conditions, but progress requires characterization of the DSE under conditions that are relevant for folding and aggregation, i.e., under conditions where the native state is stable. Comparative experimental studies of a set of three representative model proteins will be carried out under physiologically relevant designed conditions. The experimental work will be closely integrated with efforts by theoretical collaborators. A range of educational and outreach activities are planned. An 'adopt a lab' program has been initiated as part of the first year undergraduate honors lab class and will be further developed. Interactions with Stony Brook University?s project WISE (Women in Science and Engineering) program will be established to provide support and encouragement to undergraduate women who are interested in science and engineering. Laboratory exercises will also be designed to interface with project WISE. Graduate and undergraduate training and mentoring are key components of the project. Outreach activities include visits and seminars at undergraduate institutions, as well as collaborations with researchers at primarily undergraduate institutions. The research goals of the project aim to answer fundamental questions in protein folding. As part of the work, new tools and methodologies will also be developed which can be applied to a range of problems in protein structure and dynamics beyond the area of protein folding.