title: Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan.
abstract: The B1 domain of Streptococcal protein G (GB1) is a small, thermostable protein containing a single tryptophan residue. We recorded time-resolved fluorescence of the wild-type GB1 and its 5-fluorotryptophan (5FTrp) variant at more than 30 emission wavelengths between 300 and 470 nm. The time-resolved emission spectra reveal no signs of heterogeneity, but show a time-dependent red shift characteristic of microscopic dielectric relaxation. This is true for both 5FTrp and unmodified Trp in GB1. The time-dependent red shifts in the fluorescence of 5FTrp and unmodified Trp are essentially identical, confirming that the shift is caused by the relaxation of the protein matrix rather than by the fluorophore itself. The total amplitude (but not the rate) of the time-dependent red shift depends on the fluorophore, specifically, on the magnitude of the vector difference between its excited state and ground state electric dipole moments; for 5FTrp this is estimated to be about 88% of that for the unmodified Trp. The decay of the excited state fluorophore population is not monoexponential for either fluorophore; however, the deviation from the monoexponential decay law is larger in the case of unmodified Trp. The relaxation dynamics of GB1 was found to be considerably faster than that of other proteins studied previously, consistent with the small size, tightly packed core, and high thermodynamic stability of GB1.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Toptygin
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.forename: Ludwig
authors.initials: L
authors.lastname: Brand
mesh_terms: D036103:Nanotechnology; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; C026927:5-fluorotryptophan; D014364:Tryptophan