Protein compactness measured by fluorescence resonance energy transfer - Human carbonic anhydrase II Is considerably expanded by the interaction of GroEL

Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylami no)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence i n fluorescence resonance energy transfer. The ratio between the fluorescenc e intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid ( AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nn (direct AEDANS absorption) was used to estimate the average distances between the seven T rp residues in human carbonic anhydrase II (HCA II) and the AEDANS label, G uanidine HCl denaturation of the HCA II variants was also performed to obta in a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the prot ein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL, It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA I I to bind to GroEL, and the volume of the molten globule intermediate incre ased similar to2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (t o similar to 117,000 Angstrom (3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity, Recently, we f ound that HCA II binding causes such an inflation of the GroEL molecule, an d this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarstrom, P., Persson, M., Owenius, R., Li ndgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), there by facilitating rearrangement of misfolded structure.