Figure 1.
The localizations of chromophore (green) and single tryptophan residue Trp 57 (red) are shown. Six amino acids which were mutated in the 'cycle-3' GFP variant to yield sfGFP (S30R, Y39N, N105T, Y145F, I171V and A206V) are drawn as blue stick unions. The amino acids corresponding to the 'cycle-3' mutations F99S, M153T and V163A are shown as yellow stick unions. The figure is created on the basis of PDB data [38] with the files 2B3P.ent for sfGFP [35], using the graphical software VMD [69] and Raster 3D [70].
Figure 2.
Physicochemical characteristics of sfGFP (black line) in comparison with EGFP (gray line).
(A) Absorption spectra in UV- and visible spectra regions, (inset to A) CD in visible-UV region. (B) CD in far-UV and near-UV (inset to B) regions. (C) Intrinsic fluorescence spectra at excitation wavelength of 297 nm. (D) Green chromophore fluorescence of both sfGFP and EGFP at excitation wavelength of 390 nm (solid black and gray lines, respectively). Fluorescence spectrum of sfGFP at excitation wavelength of 295 nm (dotted line) is also shown. Fluorescence spectra of sfGFP at excitation wavelengths of 390 nm (solid line), 485 nm (dashed line), corresponding to absorption of neutral and anionic chromophore, are presented on inset to D.
Table 1.
Characteristics of intrinsic and chromophore fluorescence of sfGFP in native and unfolded states.
Figure 3.
The rate of sfGFP unfolding induced by different denaturing agents.
The influence of GTC (main panel) or GdnHCl (inset) was recorded by chromophore fluorescence intensity at 494 nm. . Numbers on the curves are final denaturant concentrations in protein samples. The dashed vertical indicates the time at which the values of chromophore fluorescence intensity were compared.
Figure 4.
Conformational changes of sfGFP induced by GTC.
(A) Changes in fluorescence intensity recorded at 320 nm. (B) Changes of parameter A. (C) Changes of fluorescence anisotropy at emission wavelength of 365 nm. . Red circles and line indicate unfolding and blue circles and line represent refolding. Measurements were performed after 24 h incubation of native or denatured protein in the GTC presence. The values of parameter A of sfGFP measured after incubation of native protein in the GTC presence during 1 h (gray circles and line), 45 h (black triangles), 69 h (reversed gray triangles) and 94 h (gray crosses) are also indicated.
Figure 5.
Conformational changes of sfGFP induced by GTC.
(A) Changes of visible absorption spectra at increasing of GTC concentration as shown by arrows. Numbers at the curves indicate the denaturant concentration. (B) Changes of absorbance at 390 nm (gray circles and line) and 490 nm (black circles and line). (C and D) Changes of corrected fluorescence intensity of green chromophore at two wavelengths of excitation of 365 nm and 470 nm corrected to the change of chromophore absorption spectra. Red circles and line indicate unfolding, whereas blue circles and line represent refolding. Measurements were performed after 24 h incubation of native or denatured protein in the GTC presence. The values of chromophore intensity of sfGFP measured after incubation of native protein in the GTC presence during 1 h (gray circles and line), 45 h (black triangles), 69 h (reversed gray triangles) and 94 h (gray crosses) are also indicated. Inset to panel C: experimentally recorded (curve 1, gray), corrected to total density of solution as I/W, where (curve 2, pink), and corrected to the change of chromophore absorption spectra (see, panel C) on GTC concentration (curve 3, red) [47], [48], [49].
Figure 6.
Changes of hydrodynamic dimensions of sfGFP induced by GTC.
(A) Changes of elution profile of sfGFP at increasing denaturant concentration. Numerals at the curves specify applied denaturant concentration. (B) Changes of the position of elution peaks of compact and denatured molecules (red and blue circles, respectively) and the change of averaged elution volume of sfGFP (black triangles). The value of averaged elution volume (<V>) was calculated as , were fc(d) is portion of compact (denatured) molecules and Vc(d) is elution volume of molecules in these states. The value of fc(d) is estimated as
, were Sc(d) represents the area under peak corresponding to compact (denatured) molecules.
Figure 7.
Kinetics of GdnHCl-induced unfolding of EGFP (A) and sfGFP (B).
The changes of chromophore fluorescence intensity at 494 nm during first 10 min of protein unfolding are shown. . Numbers at the curves indicate the denaturant concentration.
Figure 8.
The structure of chromophore environment of sfGFP (A) and EGFP (B).
The localizations of residues involved in chromophore environment are shown. Tyr145 near the chromophore of EGFP (B), which is absent in the environment of sfGFP (A), is drawn in a distinct manner. Carbon, nitrogen and oxygen are gray, blue and red, respectively. The figure is created on the basis of PDB data [38] with the files 2B3P.ent for sfGFP [35] and 2Y0G.ent for EGFP [39], using the graphical software VMD [69] and Raster 3D [70].
Table 2.
Side chain conformation of Trp57 in sfGFP.
Table 3.
Characteristics of the Trp57 microenvironment in sfGFP.