High-resolution probing of local conformational changes in proteins by theuse of multiple labeling: Unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iod oacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methan ethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoac etyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoace tyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 i n human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an in termediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial cha nge in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less f lexible labels BADAN and MTSSL revealed less pronounced spectroscopic chang es in the native-to-intermediate transition, 6.6-9.0 Angstrom from the back bone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of s oluble but irreversibly aggregated oligomeric protein was identified from r efolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-sha pe simulations together with fluorescence lifetime and anisotropy measureme nts provided a picture of the self-assembled protein as a disordered protei n structure with a representation of both compact as well as dynamic and po lar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfold ing and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.