Folding pathways of immunoglobulin domains. The folding kinetics of the C.gamma.3 domain of human IgG1

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Abstract
The in vitro folding kinetics of a fragment corresponding to an intact dimer of the C.gamma.3 domain of human Ig[immunoglobulin]G1 (pFc'') were monitored via the large changes in tryptophan fluorescence which accompany these processes. In going from the guanidine hydrochloride (Gdn .cntdot. HCl) induced unfolded state (4.0 M Gdn .cntdot. HCl) to the native state (0.5 M Gdn .cntdot. HCL), 3 well-separated, 1st-order processes were observed, having time constants of 5, 50 and 350 s and roughly equal amplitudes. These values were concentration independent, a fact consistent with their being no fluorescence change accompanying dimerization. These time constants are 1-2 orders of magnitude slower than those observed for proteins of similar size such as RNase or cytochrome c, most probably reflecting the complex processes involved in forming the correct .beta.-sheet arrangement of Ig domains. The corresponding unfolding transition is biphasic, having time constant values of 50 and 500 s, the latter comprising 80% of the fluorescence change. These data indicate the presence of at least 1 species with intermediate fluorescence along the unfolding pathway. Gdn .cntdot. HCl concentration jumps were also performed over various intervals within the transition zone. The results are not consistent with a fully reversible mechanism. In the absence of the intrachain disulfide bond, pFc'' exists in an unfolded state even at 0.5 M Gdn .cntdot. HCl. In a concomitant refolding and reoxidation experiment (at 0.5 M Gdn .cntdot. HCl and using an optimal disulfide interchange catalytic system), the time constant for disulfide formation was in the range of 80-200 s and the fluorescence change revealed a lag phase analyzable in terms of rate-limiting reoxidation and refolding times consistent with those observed for the initially disulfide bonded species. Under similar conditions, but at 4 M Gdn .cntdot. HCl, reoxidation was more than 2 orders of magnitude slower, suggesting that reoxidation is directed by a refolding nucleation event.