CHARACTERIZATION OF A FOLDING INTERMEDIATE OF HUMAN CARBONIC-ANHYDRASE-II - PROBING LOCAL MOBILITY BY ELECTRON-PARAMAGNETIC-RESONANCE

The spin-labeling method was used to investigate human carbonic anhydr ase, HCA II, undergoing unfolding induced by guanidine-HCl (Gu-HCl). T he spin-probe, etramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzym e. The electron paramagnetic resonance spectrum of the folded structur e showed the characteristic slow motional spectra. When the concentrat ion of the denaturing agent, Gu-HCl, was gradually increased, new spec tral components with narrower lines evolved to give complex electron p aramagnetic resonance spectra, apparently containing superimposed cont ributions from several components of different mobility. By a differen tiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCl concentration, The ampli tude of difference spectra versus Gu-HCI concentration showed two dist inct maxima, indicating the existence of a folding intermediate state/ structure. The results were found to agree with optical absorption dat a, which showed similar transitions at the same Gu-HCl concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, foldi ng intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCl were obtained by least squares fitting of the simulated spectra to the exp erimental ones. The folding intermediate was found to have a maximum p opulation of 39 +/- 4% at similar to 0.7 M Gu-HCl.