The effect of pH and ionic strength on sfGFP spectroscopic characteristics.

<p>Absorption spectra (<b><i>A</i></b>) and fluorescence spectra (<b><i>B</i></b>) of sfGFP in the solutions with different pH. Fluorescence was excited at 390 nm and normalized to total fluorescence at current pH value. The numbers on panels A and B are the values of pH in solution. Absorption spectra (<b><i>C</i></b>) and fluorescence spectra (<b><i>D</i></b>) of sfGFP in the buffered solution and in solutions with the equal ionic strength but containing 0.7 M Na₂SO₄ or 2.1 M NaCl. Fluorescence was excited at 390 nm and normalized in the same manner as data on panel B.</p

Molar absorption spectra of neutral (pink), anionic (red) and complexed with Cl⁻ (green) states of sfGFP.

<p>Spectra decomposition was made on the basis of absorption spectra of sfGFP in the presence of studied agents. For details see the discussion section.</p

The effect of ionic denaturants and salts on the CD in the visible range of sfGFP.

<p>(<b><i>A</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final GTC concentrations of 0.0 (black line), 0.04 (red line), 0.2 (green line), and 0.5 M (blue line). (<b><i>B</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final NaSCN concentrations of 0.0 (black line), 0.05 (red line), 0.2 (green line), and 0.5 M (blue line). (<b><i>C</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final GdnHCl concentrations of 0.0 (black line), 0.25 (red line), 0.5 (green line), 2.0 M (blue line) and 2.5 M (pink line). (<b><i>D</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final NaCl concentrations of 0.0 (black line), 1.0 (red line), 1.25 (green line), 2.0 M (blue line) and 2.5 M (pink line).</p

Tertiary structure changes for bOBP in different structural states are indicated by intrinsic tryptophan fluorescence (<i>A</i>, λ_ex = 297 nm) and near-UV CD (<i>B</i>).

<p>The spectra shown are for bOBP with GdnHCl at various concentrations: 0.0 (black solid line), 0.5 (blue solid line), 1.6 (red solid line) and 3.0 M (black dotted line).</p

bOBP spatial pattern in two projections.

<p>The individual subunits in the protein are in gray and blue. The tryptophan residues in the different subunits are indicated in red and magenta. The conserved residue Tyr 83 is a gate for ligands <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085169#pone.0085169-Golebiowski1" target="_blank">[7]</a> and is shown in green. The drawing was generated based on the 1OBP file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085169#pone.0085169-Tegoni1" target="_blank">[29]</a> from PDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085169#pone.0085169-Dutta1" target="_blank">[28]</a> using the graphic software VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085169#pone.0085169-Hsin1" target="_blank">[52]</a> and Raster3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085169#pone.0085169-Merritt1" target="_blank">[53]</a>.</p

The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

<div><p>A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer’s β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.</p></div

Physicochemical characteristics of sfGFP (black line) in comparison with EGFP (gray line).

<p>(<b><i>A</i></b>) Absorption spectra in UV- and visible spectra regions, (inset to <b><i>A</i></b>) CD in visible-UV region. (<b><i>B</i></b>) CD in far-UV and near-UV (inset to <b><i>B</i></b>) regions. (<b><i>C</i></b>) Intrinsic fluorescence spectra at excitation wavelength of 297 nm. (<b><i>D</i></b>) 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 <b><i>D</i></b>.</p

bOBP conformational changes induced by GdnHCl.

<p><b><i>A</i></b>: Changes in parameter <i>A</i>, λ_ex = 297 nm; <b><i>B</i></b>: changes in fluorescence anisotropy at the emission wavelength 365 nm, λ_ex = 297 nm; <b><i>C</i></b>: changes in the fluorescence lifetime <i>τ</i>, λ_ex = 297 nm and λ_em = 335 nm; <b><i>D</i></b>: changes in fluorescence intensity at 320 nm, λ_ex = 297 nm; <b><i>E</i></b>: changes the in fluorescence intensity at 365 nm, λ_ex = 297 nm; and <b><i>F</i></b>: changes in the ellipticity at 222 nm. CD spectrum in the far-UV region for bOBP in buffered solution (insert for <b><i>E</i></b>). The measurements were preceded by incubating the protein in a solution with the appropriate GdnHCl concentration at 4°C for 2 (gray crosses), 24 (red circles) and 48 h (brown crosses). The open symbols indicate unfolding, whereas the closed symbols represent refolding.</p

Kinetics of GdnHCl-induced unfolding of EGFP (<i>A</i>) and sfGFP (<i>B</i>).

<p>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.</p

Parametric dependencies between the fluorescence intensities at 320 and 365-induced bOBP unfolding processes.

<p>The excitation wavelength is at 297–0.5 (blue), 0.5–1.6 (red) and greater than 1.6 (green).</p