Solvent-Induced Protein Refolding at Low Temperatures

Protein refolding at low temperatures is shown for a self-assembled system of human serum albumin (HSA) and spin-labeled fatty acids (FAs), in ternary solvent mixtures with usually denaturing cosolvents ethanol or ionic liquids (ILs). When HSA is natively folded, it offers FA binding sites, and the uptake and the distribution of these FA binding pockets have characteristic continuous wave electron paramagnetic resonance (CW EPR) and double electron–electron resonance (DEER) signatures. At room temperature, CW EPR shows that the addition of 35% (v/v) of ethanol or IL leads to HSA being unfolded. A temperature decrease yields bimodal CW EPR spectra with bound FA and free FA signals, indicating at least partial refolding of HSA, which is also confirmed by corresponding DEER data. This finding is based on increased protein stability at lower temperatures and a change in the preferential solvation of the protein by glycerol in the ternary solvent mixtures

Intramolecular part of the DEER time-domain data.

<p>Extracted distance distributions of 5-DSA (<i>A</i>, <i>B</i>) and 16-DSA (<i>C</i>, <i>D</i>) in BSA solutions with varying numbers of paramagnetic and diamagnetic FAs (the numbers denote: Albumin : DSA : rDSA). Δ indicates the modulation depths.</p

Comparison of the distance distributions of fatty acids.

<p>5-DSA (<i>A</i>) and 16-DSA (<i>B</i>) obtained from DEER measurements in HSA (red, 1∶2:4) and in BSA (black, 1∶2:5) solutions with the calculated distributions from the crystal structure of HSA (blue) assuming that all seven binding sites are occupied by FAs (Albumin:DSA:rDSA).</p

Site 5.

<p>(<i>A</i>) Site 5 in the subdomain of IIIB in HSA with bound stearic acid (pdb-ID: 1e7i). Identical amino acids in HSA and BSA are blue, differing amino acids are red. (<i>B</i>) Plot of ΔHI for residues 498–509 and for 560–580.</p

Loop region.

<p>(<i>A</i>) Subdomain IIB of HSA with bound stearic acids (pdb-ID: 1e7i). Identical amino acids in HSA and BSA are blue, differing amino acids are red. (<i>B</i>) Plot of ΔHI for residues 297–320 and for 351–380.</p

Location of fatty acids and their paramagnetic centers.

<p>Overview of the occupation and location of binding pockets of HSA with seven 5-DSA and 16-DSA ligands (PDB ID: 1e7i <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045681#pone.0045681-Bhattacharya1" target="_blank">[27]</a>). (<i>A</i>) congruent molecular surface area of HSA (bright blue) and FA_is (brown). (<i>B</i>) FA_is with according paramagnetic centers of 16-DSA (pc_i) in red. (<i>C</i>) paramagnetic centers (pc_i) of 5-DSA with their ensuing 21 possible interspin distances indicated (blue lines). (<i>D</i>) paramagnetic centers (pc_i) of 16-DSA with their ensuing 21 possible interspin distances indicated (blue lines).</p

Crystal structure alignment of HSA and BSA.

<p>HSA without FAs (pdb-ID: 1BM0, blue) aligned with the crystal structure of BSA without FAs (pdb-ID: 3v03, red) using the MUSTANG algorithm. The regions of interest (site 1, site 5, intersection, loop) are highlighted in green.</p

Modeling Excluded Volume Effects for the Faithful Description of the Background Signal in Double Electron–Electron Resonance

We discuss excluded volume effects on the background signal of double electron–electron resonance (DEER) experiments. Assuming spherically symmetric pervaded volumes, an analytical expression of the background signal is derived based on the shell-factorization approach. The effects of crowding and off-center label positions are discussed. Crowding is taken into account using the Percus–Yevick approximation for the radial distribution function of the particle centers. In addition, a versatile approach relating the pair-correlation function of the particle centers with those of off-center labels is introduced. Limiting expressions applying to short and long dipolar evolution times are derived. Furthermore, we show under which conditions the background with significant excluded volume effects resembles that originating from a fractal dimensionality ranging from 3 to 6. DEER time domain data of spin-probed samples of human serum albumin (HSA) are shown to be strongly affected by excluded-volume effects. The excluded volume is determined from the simultaneous analysis of spectra recorded at various protein concentrations but a constant probe-to-protein ratio. The spin-probes 5-DOXYL-stearic acid (5-DSA) and 16-DOXYL-stearic acid (16-DSA) are used, which, when taken up by HSA, give rise to broad and well-defined distance distributions, respectively. We compare different, model-free approaches of analyzing these data. The most promising results are obtained by the concurrent Tikhonov regularization of all spectra when a common background model is simultaneously adjusted such that the a posteriori probability is maximized. For the samples of 16-DSA in HSA, this is the only approach that allows suppressing a background artifact. We suggest that the delineated simultaneous analysis procedure can be generally applied to reduce ambiguities related to the ill-posed extraction of distance distributions from DEER spectra. This approach is particularly valuable for dipolar signals resulting from broad distance distributions, which as a consequence, are devoid of explicit dipolar oscillations

Intersection region.

<p>(<i>A</i>) Intersection region between subdomains IB and IIIA of HSA with bound stearic acid (pdb-ID: 1e7i). Identical amino acids in HSA and BSA are blue, differing amino acids are red. (<i>B</i>) Plot of ΔHI for residues 180–200 and for 452–460.</p

Heme Binding Constricts the Conformational Dynamics of the Cytochrome <i>b</i>₅₅₉′ Heme Binding Cavity

Cytochrome <i>b</i>₅₅₉′ is a transmembrane protein formed by homodimerization of the 44-residue PsbF polypeptide and noncovalent binding of a heme cofactor. The PsbF polypeptide can dimerize in the absence and presence of heme. To monitor structural alterations associated with binding of heme to the apo-cytochrome, we analyzed the apo- and holo-cytochrome structure by electron paramagnetic resonance spectroscopy. Spin labeling of amino acids located close to the heme binding domain of the cytochrome revealed that the structure of the heme binding domain is unconstrained in the absence of heme. Heme binding restricts the conformational dynamics of the heme binding domain, resulting in the structurally more constricted holo-cytochrome structure