title: Motional dynamics of a buried tryptophan reveals the presence of partially structured forms during denaturation of barstar.
abstract: A double mutant of the single-domain protein barstar having a single tryptophan (W53) was made by mutating the remaining two tryptophans (W38 and W44) into phenylalanines. W53 is buried in the core of barstar. Time-resolved fluorescence of the mutant barstar (W38FW44F) showed that W53 has a single fluorescence lifetime in the native (N) state and has three lifetimes in the molten globule-like low-pH (A) form. Quenching of fluorescence by either KI or acrylamide showed that W53 is solvent inaccessible in the N-state and fairly accessible in the A-form. The denaturation of W38FW44F by guanidine hydrochloride (GdnHCI) was monitored by several probes: near-UV and far-UV circular dichroism (CD), fluorescence intensity, and steady-state and time-resolved fluorescence anisotropy. While the unfolding transitions observed through CD and fluorescence intensity coincided with each other (midpoint approximately 1.8 M GdnHCI), the transition observed through the steady-state fluorescence anisotropy was markedly different from others. Initially, the anisotropy increased with the increase in the concentration of GdnHCI and decreased subsequently. The midpoint of this titration was 2.2 M GdnHCI. Picosecond time-resolved fluorescence anisotropy showed that W38FW44F has a single rotational correlation time of 4.1 ns in the native (N) state and 1.5 as in the unfoled (U) state (6 M GdnHCI). These could be explained as being due to the absence of motional freedom of W53 in the N-state and the presence of rotational freedom in the U-state. In the intermediate concentration region (1.8-3.0 M GdnHCI), the anisotropy decays showed at least two coorrelation times, approximately 1 and 6-12 ns. These two correlation times are ascribed to partially structured forms leading to hindered rotation of W53. Thus, the usefulness of time-resolved fluorescence anisotropy in detecting partially folded structures is demonstrated.
authors.affiliation: Chemical Physics Group, Tata Institute of Fundamental Research, Bombay, India.
authors.forename: R
authors.initials: R
authors.lastname: Swaminathan
authors.forename: U
authors.initials: U
authors.lastname: Nath
authors.forename: J B
authors.initials: JB
authors.lastname: Udgaonkar
authors.forename: N
authors.initials: N
authors.lastname: Periasamy
authors.forename: G
authors.initials: G
authors.lastname: Krishnamoorthy
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006146:Guanidines; C057438:barstar protein, Bacillus amyloliquefaciens; D014364:Tryptophan; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D019791:Guanidine