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In this study, self-assembled systems of human serum albumin (HSA) and spin-labeled fatty acids are characterized by double electron-electron resonance (DEER). HSA, being the most important transport protein of the human blood, is capable to host up to seven paramagnetic fatty acid derivatives. DEER measurements of these self-assembled multispin clusters are strongly affected by correlations of more than two spins, the evaluation of the latter constituting the central topic of this paper. While the DEER modulation depth can be used to obtain qualitative information of the number of coupled spins, the quantitative analysis is hampered by the occurrence of cluster mixtures with different numbers of coupled spins and contributions from unbound spin-labeled material. Applying flip angle dependent DEER measurements, unwanted multispin correlations were found to lead not only to a broadening of the distance peaks but also to cause small distances to be overestimated and large distances to be suppressed. It is thus favorable to use spin-diluted systems with an average of two paramagnetic molecules per spin cluster when a quantitative analysis of the distance distribution is sought., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The structure of human serum albumin loaded with a metal porphyrin and fatty acids in solution is characterized by orientation-selective double electron-electron resonance (DEER) spectroscopy. Human serum albumin, spin-labeled fatty acids, and Cu(II) protoporphyrin IX-a hemin analog-form a fully self-assembled system that allows obtaining distances and mutual orientations between the paramagnetic guest molecules. We report a simplified analysis for the orientation-selective DEER data which can be applied when the orientation selection of one spin in the spin pair dominates the orientation selection of the other spin. The dipolar spectra reveal a dominant distance of 3.85 nm and a dominant orientation of the spin-spin vectors between Cu(II) protoporphyrin IX and 16-doxyl stearic acid, the electron paramagnetic resonance reporter group of the latter being located near the entry points to the fatty acid binding sites. This observation is in contrast to crystallographic data that suggest an asymmetric distribution of the entry points in the protein and hence the occurrence of various distances. In conjunction with the findings of a recent DEER study, the obtained data are indicative of a symmetric distribution of the binding site entries on the protein's surface. The overall anisotropic shape of the protein is reflected by one spin-spin vector orientation dominating the DEER data., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The effect of several ionic liquids (ILs) on the solution structure of human serum albumin (HSA) is revealed by continuous wave electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron-electron resonance (DEER) spectroscopy. HSA, the most abundant protein in human blood, is able to bind and transport multiple fatty acids (FAs). Using spin-labeled FA, the uptake of the FA by the protein and their spatial distribution in the protein can be monitored. The FA distribution provides an indirect yet effective way to characterize the structure of the protein in solution. Addition of imidazolium-based ILs to an aqueous solution of HSA/FA conjugates is accompanied by significant destabilization and unfolding of the protein's tertiary structure. In contrast, HSA maintains its tertiary structure when choline dihydrogenphosphate (dhp) is added. The comparison of FA distance distributions in HSA with and without choline dhp surprisingly revealed that with this IL, the FA anchoring units are in better agreement with the crystallographic data. Furthermore, the FA entry point distribution appears widened and more asymmetric than in pure buffer. These results indicate that choline dhp as a cosolvent may selectively stabilize HSA conformations closer to the crystal structure out of the overall conformational ensemble.