Understanding the fundamental molecular mechanisms by which proteins fold remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is contained within the amino acid sequence, but just how that """"""""code"""""""" is translated into folding pathways and the unique three-dimensional structure required for biological activity remains poorly understood. There is an urgent need for direct experimental characterization of folding intermediates to elucidate the fundamental molecular interactions that stabilize their structures and influence the rates by which they are formed and by which they progress to the native state - or to misfolded states. Since unfolded and partially folded states are implicated in the lifecycle of proteins within a cell and in the development of amyloid disease, there is also a need to characterize weakly populated intermediates formed by spontaneous unfolding of native proteins. The overall objective of the proposed research is to address these outstanding issues through kinetic and equilibrium studies of intermediates that populate the folding energy landscape of apomyoglobin. Apomyoglobin is a paradigm for protein folding, which provides unique opportunities for detailed investigations of protein folding mechanisms since it exhibits relatively straightforward folding kinetics with well-defined intermediates and forms an equilibrium molten globule, similar in structure to the kinetic intermediate, under conditions that allow detailed NMR analysis. Recently developed NMR relaxation dispersion experiments will be used to obtain novel insights into the kinetics and energetics of the U -1 and N -1 transitions, and to determine the structure of the intermediates at a level of detail that cannot be attained through conventional approaches. Experiments during the previous grant period have generated preliminary structural models of the apomyoglobin burst phase intermediate and hypotheses about the role of non-native helix packing as a kinetic trap that slows folding. The molecular origins of the non-native structure and its role in determining subsequent folding events will be investigated by mutagenesis and kinetic measurements. Efficient and error-free folding of proteins within the cell is essential to life; misfolded proteins have a high tendency to aggregate and are associated with numerous debilitating amyloid diseases including late-onset diabetes, prion disease, and Alzheimer's and Parkinson's disease. This research will advance our understanding of the mechanisms by which proteins fold correctly, and provide new insights into factors that cause them to misfold and form insoluble aggregates. ? ? ?
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