Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR.
The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.
Bann
James G
JG
Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Frieden
Carl
C
D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005461:Fluorine / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D014508:Urea / Q000737:chemistry; D010135:p-Fluorophenylalanine / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C084330:PapD protein, E coli; D010446:Peptide Fragments; D033903:Periplasmic Proteins; D005461:Fluorine; D010649:Phenylalanine; D010135:p-Fluorophenylalanine; D014508:Urea; D011392:Proline