Mapping the Folding Intermediate of Human Carbonic Anhydrase II. Probing Substructure by Chemical Reactivity and Spin and Fluorescence Labeling of Engineered Cysteine Residues

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Abstract
Several conformation-sensitive parameters have shown that human carbonic anhydrase II exists as a stable and compact equilibrium folding intermediate of molten globule type. In this study we have continued a previously initiated mapping of the intermediate structure. Cys residues were engineered, one at a time, into various regions of the protein structure, so as to obtain chemically reactive probes and handles for spectroscopic probes. These probes were used to specifically report on conformational changes accompanying the folding process. Thus, the accessibility of the introduced Cys residues to specific chemical labeling by radioactive iodoacetate was used to monitor the stability and compactness of the substructure surrounding each Cys residue. In addition, a spin-label (nitroxide radical) and a fluorescent probe (IAEDANS) were attached to the inserted SH-groups to give complementary information. The mobility of the spin-label was used to indicate local changes in structure, and the fluorophore was used to probe local changes in polarity at various stages of unfolding. Much of the predominant beta-structure, consisting of 10 beta-strands extending throughout the entire molecule, appears to be compact and largely intact in the intermediate. Thus, beta-strands 3-7, probed at positions 68, 97, 118, 123, 206, and 245, seem to have a native-like structure in the folding intermediate. In contrast, a more flexible structure is found around positions 56, 176, and 256 in the peripheral beta-strands 1, 2, and 9, showing that the stability of the secondary structure in the intermediate state is less in the outer parts of the protein. A hydrophobic region, containing beta-strands 3-5, seems to be remarkably stable and is not ruptured until strong denaturing conditions (5 M GuHCl) are applied. The stability of this hydrophobic beta-core appears to increase toward the center. This stable region is contained in the middle of a sequentially continuous antiparallel structure that spans beta-strands 2-6, suggesting that this part might represent a site where folding is initiated.

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