We plan to study the structure of members of the cyclophilin protein family in solution by NMR. These proteins have peptidyl prolyl cis trans isomerase (PPIase) activity and catalyze protein folding. Eukaryotic cyclophilins have high binding affinity for the immunosuppressant drug Cyclosporin A (CsA) and are thought to be the intracellular target. E. coli cyclophilin has much lower affinity for CsA but we have constructed an F112W Eco Cyp mutant that has 25-fold greater affinity for drug. We will start by NMR analysis of the 168 residue wild type and mutuant E. coli recombinant cyclophilins and then proceed to a human cyclophilin isoform human Cyp B (188aa), which we have recently cloned, sequenced and purified and find in the ER and golgi compartment rather than the cytoplasm, the site of human isoform A. The cyclophilins will be prepared with 15N and 13C-isotope labeling, and studies with bound 13C-cyclosporin A are planned to determine ligand-receptor interactions. We will also conduct enzyme mechanistic studies of the PPIase activity, including mutagenesis, and we will search for specific proteins and substrates.

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National Institute of General Medical Sciences (NIGMS)
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