Our general goal is to understand how the """"""""random coiled"""""""" carbonic anhydrase folds into a unique functional conformation, and the role of its metal cofactor, Zn(II), in this folding process. We plan to investigate the following specific areas: (1) Detailed multi-parameter fast kinetic studies on the refolding of the protein by stopped-flow circular dichroism, stopped-flow spectrophotometry, and possibly laser Raman spectroscopy: Multiple unfolded states will be identified and accounted in the kinetic analyses. """"""""Double-jump"""""""" and low temperature experiments will also be performed to examine the possible existence of the slow cis-trans proline isomerization. (2) Conformation and folding of active proteolytic fragments(s) of carbonic anhydrase: Fragments of the active single-nicked enzyme from limited Nargase digestion will be separated and purified. Another approach will be to sequentially """"""""trim"""""""" away the N-terminal, and/or the C-terminal segment by specific proteases to obtain active """"""""core"""""""" fragments. The conformation stability and folding kinetics of these fragments will then be studied. (3) The role of Zn(II) in the refolding of the active protein fragment(s): Equilibrium and kinetic studies followed by activity assay and several physicochemical methods, each reflecting a particular aspect of the conformation of the protein. Replacement of the Zn(II) by Co(II) will also be studied, particularly by taking advantage of the visible absorption and circular dichroism which reflects the immediate conformational environment of the metal ion. (4) The effect of solvent viscosity on the kinetics to understand the nature of segmental motions in the """"""""clustering"""""""" step(s) in protein folding. (5) Analyses of the refolding by laser Raman spectroscopy and resonance Raman spectroscopy. The results should provide fundamental knowledge of the causes of abnormality in protein structure and function, particularly those involved in cardiovascular diseases since carbonic anhydrase is an essential enzyme in respiratory function.
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| Allen, S H; Wong, K P (1986) The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes. Arch Biochem Biophys 249:137-47 |
