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.
The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.
Ionic liquids (ILs) feature a variety of properties that make them a unique class of solvents. To gain a better understanding of how ILs solvate compounds of different chemical structure, we used pulsed high-field electron paramagnetic resonance (EPR) spectroscopy at W-band (approximately 94 GHz) and continuous wave EPR at X-band (approximately 9.4 GHz) on three TEMPO-based spin probes with different substitutions at the 4-position: 4-R-2,2,6,6-tetramethylpiperidine-1-oxyl, with R = N(CH(3))(3)(+), Cat-1, R = COO(-), TEMPO-4-carboxylate, and R = OH, TEMPOL. The spin probes are dissolved in imidazolium based ILs with different alkyl chain lengths (-C(2)H(5), -C(4)H(9), -C(6)H(13)) and anions (BF(4)(-), PF(6)(-)) and also in molecular solvents (methanol, water-glycerol). X-Band EPR at RT shows that the reorientational motion of the charged spin probes in ILs is about fivefold slower than that of the TEMPOL. Moreover, anion variation from BF(4)(-) to PF(6)(-) in ILs most strongly slows down the rotational motion (as measured by the rotational correlation time tau(r)) of Cat-1, followed by TEMPOL, while tau(r) of TEMPO-4-carboxylate is least affected. The EPR parameters g(xx) and A(zz) (tensor elements of the g- and hyperfine tensor) are sensitive to environmental effects and are only fully resolved at the high field used in this study. Changes of g(xx) and A(zz) values of the Cat-1 in ILs and methanol are very small especially compared to that of TEMPO-4-carboxylate, indicating that Cat-1 is located in a polar region of the ILs resembling the situation in methanol. On the other hand, the g(xx) value of TEMPO-4-carboxylate is sensitive to the length of alkyl group which shows that TEMPO-4-carboxylate is close to the nonpolar region of ILs. The small differences in the chemical substitution of the spin probes used here are sufficient for the molecules to reside in different domains of different dielectric properties in ILs. Our combined results are in good agreement with a picture of a nanophase separation, in which the charged cations and anions form polar regions and the hydrophobic alkyl chains of the IL cations form non-polar regions.
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