The single helix in protein L is largely disrupted at the rate-limiting step in folding.

To investigate the role of helix formation in the folding of protein L, a 62 residue alpha/beta protein, we studied the consequences of both single and multiple mutations in the helix on the kinetics of folding. A triple mutant with 11 additional carbon atoms in core residues in the amino-terminal portion of the helix folded substantially faster than wild type, suggesting that hydrophobic association with residues elsewhere in the protein occurs at the rate-limiting step in folding. However, helix-destabilizing mutations had little effect on the rate of folding; in particular, a triple glycine substitution on the solvent-exposed side of the helix increased the unfolding rate 56-fold while reducing the folding rate less than threefold. Thus, in contrast to the predictions of models of folding involving the coalescence of well-formed secondary structure elements, the single helix in protein L appears to be largely disrupted at the rate-limiting step in folding and unfolding.

Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labelling.

BACKGROUND: Hydrogen exchange labelling has been a key method in characterizing the structure of transient folding intermediates. In studies of several proteins, however, there has been clear spectroscopic evidence for partial folding of some kind at very early times, before any protection from exchange was measurable. These results, presumably a consequence of limited stability of specific backbone interactions, have made it difficult to assess the extent of native-like folding in the very early intermediates. We have used a variant of the labelling method to investigate marginally stable structures formed within the first few milliseconds of refolding of two such proteins, hen lysozyme and ubiquitin. RESULTS: In lysozyme, population of a subset of native-like secondary structures on this timescale is revealed, thus reconciling the exchange behaviour with circular dichroism measurements and confirming the significance of the rapidly formed embryonic structure as a foundation for the subsequent folding pathway. In the case of ubiquitin, by contrast, no significantly protective structure was detectable, suggesting that here secondary structural elements can be populated only marginally ahead of the major cooperative folding event; this was also supported by stopped-flow circular dichroism measurements. CONCLUSIONS: The hydrogen exchange approach can be extended to probe the formation of native-like structure formed in very early folding intermediates, even when the stability of specific interactions is marginal. In the case of lysozyme, this has provided a new window on an early stage of organization of the alpha-helical domain.

Characterization of the free energy spectrum of peptostreptococcal protein L.

BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration.

Structural transitions in the protein L denatured state ensemble.

We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.