Foldase Catalyzed Protein Folding and Molecular Biology Workshops for K-12 Students

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9631485 Noiva Protein disulfide isomerase (PDI) is a "foldase" located in the lumen of the rough endoplasmic reticulum where it facilitates several of the reactions required for the folding, assembly, and posttranslational modification of secretory and membrane proteins. A site on the PDI molecule which recognizes unfolded and misfolded nascent polypeptides has been identified and demonstrated to be involved in PDI-facilitated folding. In this proposal, the role of this peptide binding site in PDI activity will be further characterized. The technique of oligonucleotide-directed site mutagenesis will be used to generate mutants of PDI lacking the peptide binding site or containing single amino acid substitutions in that site. Wild type and mutant PDI will be expressed in both E. coli and Pichia pastoris expression systems to generate the highest yield of enzyme. The effect of loss of peptide binding activity on PDI activity will be assessed on both the activity of the soluble enzyme and its ability to catalyze properly folded proteins in dog pancreas microsomes. Preliminary evidence indicates that PDI has little or no substrate sequence preference, but is hypothesized to recognize a stretch of 6 amino acids in the polypeptide chain. Substrate preferences for the PDI foldase activity will be further characterized by determining length and sequence requirements for peptide binding by PDI using a combination of site-directed mutagenesis, affinity chromatography, and affinity panning techniques. The role of calcium in PDI function in the cell will also be assessed. Calcium does not have any direct effect on PDI-facilitated folding or peptide binding activity in vitro, but may be involved in localization of PDI to the lumen of the rough endoplasmic reticulum. This calcium-mediated localization of PDI will be further characterized using the techniques of pulse-chase labeling, western blotting, and confocal microscopy. A series of summer workshops in Modern Techniques in Experimental Biology for K-12 students and teachers will be developed. Workshops will be composed of a series of experimental modules presenting techniques used in the modern molecular biology laboratory and will be tailored to the level of junior/senior high school students. Modules will be interchanged between workshops depending on the abilities and prior experiences of the students. The workshops will use a recently completed Molecular Biology Laboratory teaching facility funded by the Howard Hughes Medical Institute. %%% Current technology allows scientists to use bacterial cultures to produce recombinant human proteins of clinical and pharmaceutical importance. However, bacteria lack chaperone and foldase proteins required to fold these mammalian proteins efficiently. In this proposal, protein disulfide isomerase (PDI), one of the foldases which assists protein folding in humans, will be studied. In order to assist proteins in folding, PDI must recognize a motif on the newly synthesized protein which identifies that protein as requiring assistance for proper folding. In this proposal, the role of protein binding by PDI in assisted folding and the motif that is recognized by PDI will be characterized. Furthermore, the role of intracellular calcium in directing PDI to the area in cells where protein folding occurs will also be studied. This information will strengthen our understanding of the cellular function of PDI and lead to the better use of PDI in improving yields in the production of recombinant human proteins in bacteria. A series of summer workshops in Modern Techniques in Experimental Biology for K-12 students and teachers will be developed. These workshops will provide opportunities for students from Native American reservations and rural communities with limited educational resources to have access to current technology used in experimental biology. Students will be encouraged to learn biological techniques at the molecular level such as electrophoresis, PCR, and cloning recombinant DNA. Experimental module s offered at the workshop will reinforce science at their own schools, present new and exciting concepts and hopefully motivate students to pursue further opportunities in Biology and the Biomedical Sciences. ***