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 guanidine hydrochloride have been investigated on the manual and stopped-flow mixing time scales. The kinetics of change of far-UV circular dichroism and of intrinsic and ANS fluorescence have been compared with changes in the quenching of fluorescence by acrylamide as a probe of the accessibility of solvent to tryptophan. The binding of ANS points to hydrophobic collapse in the very early stages of folding which take place in the burst phase. This is accompanied by regain of 60-65% of native 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 less 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 formed over the next 20 s. Quenching by acrylamide increases during this period, as the transient nonnative state is disrupted and the tryptophan residue(s) become(s) reexposed to solvent. The two slowest phases are determined by the isomerization of incorrect prolyl isomers, but double jump tryptophan fluorescence and acrylamide quenching experiments show little, if any, effect of proline isomerization on the earlier phases. Hydrophobic collapse thus occurs to a folding intermediate in which there is a nonnative element of structure which has to rearrange in the later steps of folding, resulting in a nonhierarchical folding pathway. The C-terminal W290 is suggested as being involved in the nonnative intermediate. beta-Lactamase provides further evidence for the occurrence of nonnative intermediates in protein folding.
Vanhove
M
M
Laboratoire d'Enzymologie, Université de Liège, Belgium. marc.vanhove@stjude.org
Lejeune
A
A
Guillaume
G
G
Virden
R
R
Pain
R H
RH
Schmid
F X
FX
Frère
J M
JM
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D005454:Fluorescence Polarization; D007700:Kinetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D001618:beta-Lactamases / Q000737:chemistry*
D000178:Acrylamides; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1