Folding, Misfolding and Inclusions of Complex Proteins. The rules through which amino acid sequences direct the folding of newly synthesized polypeptide chains into beta sheet and parallel beta coil proteins remain obscure. The failure of such proteins to fold into the native state, and their accumulation as inactive aggregated inclusion bodies is a major problem in biomedical laboratories and in the biotechnology industry. More recently protein folding defects and protein aggregation processes have emerged as the basis of a number of human amyloid and other protein deposition diseases. Inclusion body states are only very poorly understood compared to the native states of participating chains. Over the past period of GM17,980 we have developed genetic tools and biochemical methodologies for accumulating, fractionating and probing partially folded intermediates in protein misfolding and inclusion body formation in vivo and in vitro. These developments took advantage of features of phage infected cells and phage structural proteins. Particularly useful is the parallel beta coil P22 tailspike trimer whose folding and aggregation intermediates have been identified both in vivo and in vitro. In the next period we plan to concentrate our efforts on a) identifying the buried stack residues which may be controlling parallel beta coiled folding, b) elucidating the intermediates at the junction between productive folding and misfolding, c) identifying the reactive cysteine residues involved in the formation of the transient but essential interchain S-S bonds found in tailspike protrimer intermediates, d) identifying the domains involved in polymerization of partially folded intermediates into inclusion bodies for both wild type and mutant chains; e) exploring the mechanism by which global suppressors inhibit inclusion body formation, including their potential role as chaperonin recruitment signals, f) probing the conformation of ribosome bound in vivo folding intermediates characterizing using monoclonal antibodies, and g) applying these methodologies to identifying the early stages in the folding and assembly of integral membrane proteins with the photosynthetic reaction center of photosynthetic bacteria as a model system.
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