A Collapsed Intermediate with Nonnative Packing of Hydrophobic Residues in the Folding of TEM-1 β-Lactamase

Abstract

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.

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

“…1 Such a favorable role of non-native interaction in the formation of protein folding nucleus is in line with previous experimental and theoretical studies, suggesting that specific non-native interactions may accelerate folding by reducing conformational search to the native state. [53][54][55][56][57][58][59][60][61][62][63] The identification of a critical salt-bridge in a crystallin folding/unfolding intermediate is another example of non-native interactions crucial for protein folding. Similarly, Presta and Rose 59 described helix stop signals that were not necessarily retained in the native state of the folded proteins.…”

Direct Kinetic Evidence for Folding via a Highly Compact, Misfolded State

“…This is not the first evidence for nonnative structure in folding intermediates. In the dead time intermediate of ␤-lactamase folding, indole groups are shown to be more buried than in the native state (32), and nonnative ␣-helical structure has been seen in the early folding intermediate of ␤-lactoglobulin (31). Such observations can be argued to reflect the balance between the influence of intrinsic secondary structure preferences in the first stage of folding and tertiary interactions in determining the final folded structure (31,37).…”

“…Folding of the intertwined dimeric DNA binding domain of the human papillomavirus E2 protein (29) and of the tryptophan repressor from Escherichia coli (30) is proposed to involve a nonnative, monomeric intermediate. Interestingly, the predominantly ␤-sheet protein, ␤-lactoglobulin, contains nonnative ␣-helical structure (31) in its early folding intermediate, while TEM-1 ␤-lactamase folds through a collapsed intermediate containing nonnative hydrophobic interactions (32).…”

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and19F NMR

“…The difficulty arises both from the property of the intermediates (transient, low populated, and heterogeneous) and the lack of appropriate probes to give experimental high-resolution structural information. Hydrophobic collapses, i.e., the very early stages of intermediate formation, are usually identified indirectly by using fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding (1), photo-chemically induced dynamic nuclear polarization (CIDNP) NMR (2) and mutagenesis (3)(4)(5), and chevron analysis (6). Recently, relaxation dispersion NMR has been successfully applied to SH3 domain protein to directly characterize intermediates interconverting between folded and unfolded states (ground and excited states) on the millisecond time scale (7)(8)(9)(10).…”