Fatty Acid Triangulation in Albumins Using a Landmark Spin Label

Abstract

Several spatial correlations of up to six fatty acid (FA) binding sites in albumins were found by double electron-electron resonance (DEER). A strategy was used that combines spin-labeling and spin-probing techniques in electron paramagnetic resonance (EPR) spectroscopy. This is here achieved by introducing an additional covalent landmark spin (LS) label to the self-assembled system of EPR-active, paramagnetic stearic acid derivatives and albumins. Therefore, a cysteine specific, paramagnetic LS that was attached to the albumin surface at a unique position (Cys34) provides a fixed topological reference point for monitoring statistical ligand uptake. We propose that the determination of nanoscale distance distributions emerging between the LS and EPR-active fatty acid derivatives generally allows for the direct observation of individually occupied binding sites in solution. Essentially, several binding pockets, groups of them and evidence for ligand-induced allosteric modulation can be traced from such FA-LS interspin correlations. Experimental results were substantiated with theoretical predictions from molecular dynamics (MD) simulations. It was observed that all binding sites in an albumin ensemble may be statistically filled even at the lowest level of ligand loading. This approach generally bears the potential for mapping occupation states of individual ligand binding sites in proteins using such spinlabeled ligands.

Reversible Dimerization of Human Serum Albumin

“…Here we detect and begin to characterize reversible non-covalent HSA aggregates or “dimers” using the Pulsed Dipolar EPR Spectroscopy (PDS). The PDS technique PELDOR (also known as DEER) [ 17 , 18 ] has already been applied to HSA previously with great success to study its binding of fatty acids and its structure under extreme conditions [ 19 , 20 , 21 , 22 ]. We examine whether HSA in solution, under physiologically relevant conditions, forms significant amounts of non-covalent dimers; characterize their structure; and probe whether the monomer/dimer equilibrium or kinetics is affected by post-translational modification.…”

Spin-Label Electron Paramagnetic Resonance Spectroscopy Reveals Effects of Wastewater Filter Membrane Coated with Titanium Dioxide Nanoparticles on Bovine Serum Albumin

“…Then, the five-peak component was subtracted from the composite spectra, leaving the normal mobile plus immobile components, which were then treated with the routine technique for the two components [59]. Although we see the immobile component as observed earlier [31], it is almost undetectable; hence, the outer splittings could not be determined. (Again, it should be kept in mind that the filtrated BSA sample has a 3.3-fold lower protein concentration than the other two BSA-containing samples.)…”

“…It is important to note that the source of the fluorescence signal and the EPR of spin-labelled fatty acids are not as specific as the EPR of the uniquely unblocked Cys34, since there are two Trp residues and up to seven fatty-acid-binding pockets in BSA. Indeed, there are indications that all the binding sites in an albumin may have bound fatty acid even at the lowest levels of ligand loading [31]. In addition, Cys34 (in domain I) is relatively close to the protein surface [65,66], and, when spin-labelled, it has been reported to be more accessible to collisional interaction with a water-soluble paramagnetic relaxant than the acyl chain of non-covalently bound spin-labelled fatty acids (located in hydrophobic channels in the protein) [28].…”

Synthesis of Chiral Spin-Labeled Amino Acids

“…A pulsed electron paramagnetic resonance (EPR) technique known as double electron–electron resonance (DEER) − can measure interspin distances of electrons ranging from 15 to >100 Å and is a highly effective tool to study protein conformations. − It usually depends on incorporation of two spin labels that have a stable unpaired electron, ,,− often as a modified amino acid residue. ,, The distances between these two spin-labels can be used to triangulate conformational changes in proteins stepwise by site-directed spin-labeling of various topological sites . Another approach comprises a single spin-labeled protein residue from which ligand binding can be observed. , Using spin-labeled ligands is already well established in EPR spectroscopy, and additional information about number of binding sites and affinity can be gained . A common motif is a fully α-substituted nitroxide radical akin to TEMPO (2,2,6,6-tetramethylpiperidin-1-yl-oxy). , Such moieties can be attached by selective modification of existing amino acid residues (e.g., alkylation or disulfide bridge formation to cysteine sulfhydryl by 1 or 2 , respectively) (Figure ).…”