EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: Evidence for increased flexibility of the hydrophobic core by the interaction

Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room te mperature. To further investigate this interaction we used electron paramag netic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-la beled at selected positions. From our results it is evident that protein-pr otein interactions can be specifically mapped by site-directed spin-labelin g and EPR measurements. HCA II needs to be unfolded to about the same exten t as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer par ts of the HCA II molecule, such as peripheral beta-strands and the N-termin al domain, which have previously been shown to be rather unstable. As a res ult of the interaction, the rigid and compact hydrophobic core exhibits hig her flexibility than in the molten globule, which is likely to facilitate r earrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactio ns appear to be most important in stabilizing the GroEL-substrate complex.