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.
Scalley
M L
ML
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Nauli
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S
Gladwin
S T
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Baker
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C116593:L-protein, Peptococcus magnus; D009282:Naphthalenesulfonates; D010455:Peptides; C006421:1,5-I-AEDANS