A COLLAPSED INTERMEDIATE WITH NONNATIVE PACKING OF HYDROPHOBIC RESIDUES IN THE FOLDING OF TEM-1 BETA-LACTAMASE

The kinetics of refolding of TEM-1 beta-lactamase from solution in gua nidine hydrochloride have been investigated on the manual and stopped- flow mixing time scales. The kinetics of change of far-UV circular dic hroism and of intrinsic and ANS fluorescence have been compared with c hanges in the quenching of fluorescence by acrylamide as a probe of th e accessibility of solvent to tryptophan. The binding of ANS points to hydrophobic collapse in the very early stages of folding which take p lace in the burst phase. This is accompanied by regain of 60-65% of-na tive ellipticity, indicating formation of a significant proportion of secondary structure, Also in the burst phase, the tryptophan residues, which are largely exposed to solvent in the native protein, become le ss accessible to acrylamide, and the intrinsic fluorescence increases markedly. An early intermediate is thus formed in which tryptophan is more buried than in the native protein. Further intermediates are form ed over the next 20 s. Quenching by acrylamide increases during this p eriod, as the transient nonnative state is disrupted and the tryptopha n residue(s) become(s) reexposed to solvent, The two slowest phases ar e determined by the isomerization of incorrect prolyl isomers, but dou ble jump tryptophan fluorescence and acrylamide quenching experiments show little, if any, effect of proline isomerization on the earlier ph ases, Hydrophobic collapse thus occurs to a folding intermediate in wh ich there is a nonnative element of structure which has to rearrange i n the later steps of folding, resulting in a nonhierarchical folding p athway. The C-terminal W290 is suggested as being involved in the nonn ative intermediate. beta-Lactamase provides further evidence for the o ccurrence of nonnative intermediates in protein folding.