REFOLDING OF CARBONIC ANHYDRASE

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DESCRIPTION: An understanding of protein folding and re-folding is key to optium use of genetically engineered proteins. Previous studies have shown that when carbonic anhydrase is denatured and re-folded, catalytic activity is regained, although there appear to be some small changes. ... Many tests of refolded proteins examine overall structure and function. It is proposed to examine detailed changes at or near the catalytic site. Co(II) can be substituted for the native Zn(II) at the active site of carbonic anhydrase. It is proposed to use Co(II) as a spectroscopic probe of carbonic anhydrase before and after re-folding. Pulsed EPR techniques will be used to analyze the interaction of the Co(II) with three histidines at the metal-binding site and to examine the hydration of the metal site. Changes in electron-electron spin-spin interaction between Co(II) and a nitroxyl spin label added to the protein either as an inhibitor or covalently attached to the single cysteine residue in human carbonic anhydrase will be used to probe longer-range conformational changes. The data for the enzyme before re-folding will be compared with X-ray crystallographic data in the literature to validate the experimental methods. The techniques employed in this study should be applicable to other Zn(II)-containing proteins for which it has been shown that Co(II) can replace the Zn(II) with retention of catalytic activity ... .