Assessment of the Role of 2,2,2-Trifluoroethanol Solvent Dynamics in Inducing Conformational Transitions in Melittin: An Approach with Solvent ¹⁹F Low-Field NMR Relaxation and Overhauser Dynamic Nuclear Polarization Studies

Abstract

2,2,2-Trifluoroethanol (TFE) is one of the fluoroalcohols that have been known to induce and stabilize an open helical structure in many proteins and peptides. The current study has benchmarked low-field 19 F NMR relaxation and 19 F Overhauser dynamic nuclear polarization (ODNP) by providing a brief account of TFE solvent dynamics in a model melittin (MLT, an antimicrobial peptide) solution with a TFE−D 2 O cosolvent mixture at pH 7.4. Further, this approach has been employed to reveal the solvation of MLT by TFE in a nonbuffered solution with pH 2.8 for the first time. The structural transition of MLT has been elucidated via solvent dynamics by measuring the 19 F TFE relaxation rates at 0.34 T for various TFE−D 2 O compositions in the absence (bulk TFE) and in the presence of MLT at both the pH values. A complementary initial record of circular dichroism experiments on these aqueous MLT solutions with TFE as the cosolvent at two different pH conditions demonstrated the structural transition from a random coil to a helical or from a folded helical to an open helical structure. The molecular correlation time derived from the corresponding relaxation rates shows that TFE resides on the MLT surface in both pH conditions. However, the trends in the variation of molecular correlation time ratio as a function of TFE concentration represent that the mechanism and the extent to which TFE affects the MLT structural integrity are different at different pH values. The extraction of the DNP coupling parameter from steady-state 19 F ODNP experiments performed in the presence of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl at 0.34 T revealed changes in the solvation dynamics of TFE concomitant with the MLT structural transition. In summary, 19 F relaxation and ODNP measurements made at a low field have allowed direct monitoring of TFE dynamics during the MLT structural transition in terms of preferential solvation. The choice of experiments performed at a moderately low field (0.34 T) enabled us to exploit on the one hand almost 1200-fold mitigation of the strong contribution of 19 F chemical shift anisotropy at 11.76 T, whereas on the other hand, the ODNP experiment offered a window for probing molecular dynamics on timescales of the order of 10−1000 ps.

How well does molecular simulation reproduce environment-specific conformations of the intrinsically disordered peptides PLP, TP2 and ONEG?

“…The choice of TFE : water ($8 : 2 vol%) and chloroform : methanol : water ($4 : 4 : 1 vol%) as membranemimicking solvents was made because the use of these solvent mixtures for membrane-protein extraction, CD and/or NMR has precedent in the experimental literature. 6,[67][68][69][70] Hydrophobic solvents have also been used to mimic the low polarity of protein binding pockets for CD and NMR experiments of intrinsically disordered protein-binding peptides. 4,71 Following initial simulations, it was observed that the chloroform : methanol : water ($4 : 4 : 1 vol%) mixture separates into two phases when using the CGenFF/DH models (Fig.…”

Bioevaluation and Targeted Modification of Temporin-FL From the Skin Secretion of Dark-Spotted Frog (Pelophylax nigromaculatus)

“…The temporins adopt extended or unstructured conformers in other environments, this conformational phase changes would prevent indiscriminate membranolytic activity and lower the host cell toxicity ( Travkova et al, 2017 ). A previous study about TFE as a peptide stabilizes cosolvent indicated that TFE could help helical peptides maintain their secondary structure by promoting intramolecular hydrogen bond formation and peptides aggregation ( Chaubey et al, 2020 ), therefore reflects the mechanisms of peptide-membrane interaction when the AMPs contact with pathogenic microorganisms. This phenomenon has also been reported in previous studies of temporins ( Mangoni, 2006 ; Sang et al, 2018 ).…”

Challenges and Advances in the Application of Dynamic Nuclear Polarization to Liquid-State NMR Spectroscopy

“…Chaubey et al observed changes in the dynamics of the solvent 2,2,2-trifluoroethanol (TFE) when the peptide melittin undergoes structural reorganization from a random coil to a helical structure. They measured the DNP enhancement of 19 F by the polarizing agent TEMPOL and used the DNP coupling factor as an indicator of the correlation time of the cosolvent TFE . Hyperpolarized water has also been used to measure protein hydration by increasing the sensitivity of techniques such as fast 2D NMR, exchange spectroscopy (EXSY), and nuclear Overhauser effect spectroscopy (NOESY). − …”

“…They measured the DNP enhancement of 19 F by the polarizing agent TEMPOL and used the DNP coupling factor as an indicator of the correlation time of the cosolvent TFE. 40 Hyperpolarized water has also been used to measure protein hydration by increasing the sensitivity of techniques such as fast 2D NMR, exchange spectroscopy (EXSY), and nuclear Overhauser effect spectroscopy (NOESY). 41−44 3.2.…”