Protein folding pathways of adenylate kinase from E-coli: Hydrostatic pressure and stopped-flow studies

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enz yme with no tryptophan and a single cysteine residue. We have constructed s ix single-Trp mutants of AKe to facilitate optical studies of these protein s and to specifically examine the interrelationship between their structure , function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The nati ve structure of AKe was transformed to a non-native, yet pressure stable, c onformation by hydrostatic pressure of about 300 MPa. This pressure labilit y of AKe is rather low for a monomeric protein and presumably may be attrib uted to substantial conformational flexibility and a correspondingly large volume change The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 'C), the re folding process of pressureexposed AKe mutants displayed a significant hyst eresis. The observation of a slow refolding rate in the 193 region and a fa ster folding rate around the active site (86, 41, 73 regions) leads us to s uggest that in the folding process, priority is afforded to functional regi ons. The slow structural return of the 193 region apparently does not hinde r the more rapid return of enzymatic activity of AKe. Circular dichroism st udies on the pressure-denatured Y193W mutant show that the secondary struct ure (calculated from far-UV spectra) returned at a rapid rate. but the tert iary structure alignment (calculated from near-UV spectra) around the 193 r egion occurred more slowly at rates comparable to those detected by fluores cence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower r efolding process around the 193 region than near the active site. Fast refo lding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differen ces in regional folding rates of AKe are not derived from the specific dena turation methods, but rather are inherent in the structural organization of the protein.