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Transmembrane helix folding and self-association play important roles in biological signaling and transportation pathways across biomembranes. With molecular simulations, studies to explore the structural biochemistry of this process have been limited to focusing on individual fragments of this process – either helix formation or dimerization. While at an atomistic resolution, it can be prohibitive to access long spatio-temporal scales, at the coarse grained (CG) level, current methods either employ additional constraints to prevent spontaneous unfolding or have a low resolution on sidechain beads that restricts the study of dimer disruption caused by mutations. To address these research gaps, in this work, we apply our recent, in-house developed CG model ( ProMPT ) to study the folding and dimerization of Glycophorin A (GpA) and its mutants in the presence of Dodecyl-phosphocholine (DPC) micelles. Our results first validate the two-stage model that folding and dimerization are independent events for transmembrane helices and found a positive correlation between helix folding and DPC-peptide contacts. The wild type (WT) GpA is observed to be a right-handed dimer with specific GxxxG contacts, which agrees with experimental findings. Specific point mutations reveal several features responsible for the structural stability of GpA. While the T87L mutant forms anti-parallel dimers due to an absence of T87 interhelical hydrogen bonds, a slight loss in helicity and a hinge-like feature at the GxxxG region develops for the G79L mutant. We note that the local changes in the hydrophobic environment, affected by the point mutation, contribute to the development of this helical bend. This work presents a holistic overview of the structural stability of GpA in a micellar environment, while taking secondary structural fluctuations into account. Moreover, it presents opportunities for applications of computationally efficient CG models to study conformational alterations of transmembrane proteins that have physiological relevance. 

Long-term preservation of proteins at room temperature continues to be a major challenge. Towards using ionic liquids (ILs) to address this challenge, here we present a combination of experiments and simulations to investigate changes in lysozyme upon rehydration from IL mixtures using two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO 4 ] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et 2 PO 4 ]). Various spectroscopic experiments and molecular dynamics simulations are performed to ascertain the structure and activity of lysozyme. Circular dichroism spectroscopy confirms that lysozyme maintains its secondary structure upon rehydration, even after 295 days. Increasing the IL concentration decreases the activity of lysozyme and is ultimately quenched at sufficiently high IL concentrations, but the rehydration of lysozyme from high IL concentrations completely restores its activity. Such rehydration occurs in the most common lysozyme activity assay, but without careful attention, this effect on the IL concentration can be overlooked. From simulations we observe occupation of [EMIM + ] ions near the vicinity of the active site and the ligand-lysozyme complex is less stable in the presence of ILs, which results in the reduction of lysozyme activity. Upon rehydration, fast leaving of [EMIM + ] is observed and the availability of active site is restored. In addition, suppression of structural fluctuations is also observed when in high IL concentrations, which also explains the decrease of activity. This structure suppression is recovered after undergoing rehydration. The return of native protein structure and activity indicates that after rehydration lysozyme returns to its original state. Our results also suggest a simple route to protein recovery following extended storage. 

Ionic liquids (ILs) are gaining attention as protein stabilizers and refolding additives. However, varying degrees of success with this approach motivates the need to better understand fundamental IL-protein interactions. A combination of experiment and simulation is used to investigate the thermal unfolding of lysozyme in the presence of two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO 4 ] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et 2 PO 4 ]). Both ILs reduce lysozyme melting temperature Tm , but more gradually than strong denaturants. [EMIM][Et 2 PO 4 ] lowers lysozyme Tm more readily than [EMIM][EtSO 4 ], as well as requiring less energy to unfold the protein, as determined by the calorimetric enthalpy ΔH. Intrinsic fluorescence measurements indicate that both ILs bind to tryptophan residues in a dynamic mode, and furthermore, molecular dynamics simulations show a high density of [EMIM] + near lysozyme’s Trp62 residue. For both ILs approximately half of the [EMIM] + cations near Trp62 show perfect alignment of their respective rings. The [EMIM] + cations, having a "local" effect in binding to tryptophan,likely perturb a critically important Arg-Trp-Arg bridge through favorable π − π and cation-π interactions. Simulations show that the anions, [EtSO 4 ] - and [Et 2 PO 4 ] - , interact in a "global" manner with lysozyme, due to this protein’s strong net positive charge. The anions also determine the local distribution of ions surrounding the protein. [Et 2 PO 4 ] - is found to have a closer first coordination shell around the protein and stronger Coulomb interactions with lysozyme than [EtSO 4 ] - , which could explain why the former anion is more destabilizing. Patching of ILs to the protein surface is also observed, suggesting there is no universal IL solvent for proteins, and highlighting the complexity of the IL-protein environment.