Xin Zhao

Bacteriorhodopsin (bR) is a light-driven microbial receptor, and lysine 159 (K159) is a charged residue on the cytoplasmic (CP) side of its E-F loop. However, its conformation and function remain unknown due to fast surface dynamics. By utilizing a C-13, N-15-labeled lysine (K) as an isotope probe, we created a network of site-specific amide-I vibrational signatures (backbone carbonyl stretch) to identify the frequency contribution of the labeled residues to the amide-I excitonic band structure. Thus, the red-shifted amide-I frequency in the C-13, N-15-lysine-labeled bR (uK-bR) to the unlabeled bR (WT-bR) could be differentiated and examined by ultrafast two-dimensional vibrational echo infrared (2D IR) spectroscopy. Our results showed that the backbone carbonyl of K159 is located at a high frequency of ca. 1693 cm(-1) and has a vibrational excited-state relaxation time shorter than the bulk helical amide-I mode at the same frequency, suggesting that K159 may possess a hydrogen-bonded gamma-turn structure with E161, one of the carboxylate residues on the CP surface of bR. The 2D solid-state NMR study of uK-bR also revealed conformational dependent lysine residues, from which K159 was found to involve the turn motif. This gamma-turn structure maintained by K159 may help to stabilize the E-F loop and support E161 in attracting protons from the bulk during the late stage of the bR photocycle. The combined spectroscopic approach illustrated in this work may be applied to map residue-specific local structures and dynamics of other receptors and large proteins.

S-TGA-1 and PGP-Me are native archaeal lipids associated with the bacteriorhodopsin (bR) trimer and contribute to protein stabilization and native dynamics for proton transfer. However, little is known about the underlying molecular mechanism of how these lipids regulate bR trimerization and efficient photocycling. Here, we explored the specific binding of S-TGA-1 and PGP-Me with the bR trimer and elucidated how specific interactions modulate the bR trimeric structure and proton release and uptake using long-term atomistic molecular dynamic simulations. Our results showed that S-TGA-1 and PGP-Me are essential for stabilizing the bR trimer and maintaining the coherent conformational dynamics necessary for proton transfer. The specific binding of S-TGA-1 with W80 and K129 regulates proton release on the extracellular surface by forming a “Glu-shared” model. The interaction of PGP-Me with K40 ensures proton uptake by accommodating the conformation of the helices to recruit enough water molecules on the cytoplasmic side. The present study results could fill in the theoretical gaps of studies on the functional role of archaeal lipids and could provide a reference for other membrane proteins containing similar archaeal lipids.

Aromatic residues are highly conserved in microbial photoreceptors and play crucial roles in the dynamic regulation of receptor functions. However, little is known about the dynamic mechanism of the functional role of those highly conserved aromatic residues during the receptor photocycle. Tyrosine 185 (Y185) is a highly conserved aromatic residue within the retinal binding pocket of bacteriorhodopsin (bR). In this study, we explored the molecular mechanism of the dynamic coupling of Y185 with the bR photocycle by automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) calculations and molecular dynamic (MD) simulations based on chemical shifts obtained by 2D solid-state NMR correlation experiments. We observed that Y185 plays a significant role in regulating the retinal cis–trans thermal equilibrium, stabilizing the pentagonal H-bond network, participating in the orientation switch of Schiff Base (SB) nitrogen, and opening the F42 gate by interacting with the retinal and several key residues along the proton translocation channel. Our findings provide a detailed molecular mechanism of the dynamic couplings of Y185 and the bR photocycle from a structural perspective. The method used in this paper may be applied to the study of other microbial photoreceptors.

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin‐4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid‐state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark‐adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin‐4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid‐state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark‐adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.

In this study, we described the chain order change of binary unsaturated lipid bilayers by studying pure 2-oleoyl-1-pamlitoyl-sn-glyecro-3-phosphocholine (POPC) bilayers, 2-oleoyl-1-pamlitoyl-sn-glyecro-3-glycerol (POPG) bilayers, and POPC/POPG bilayers. The interactions between the POPC/POPG membrane and three model peptides containing basic, aromatic, and hydrophobic residues were studied to investigate the effects of the peptide orientation on the acyl chain order of the POPC/POPG bilayers. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopic studies showed that the acyl chain order of the POPC/POPG bilayers decreased as compared to that of the pure POPC or POPG bilayers. The chain order of the POPC/POPG bilayers significantly decreased when Gravin was added as compared to that when Vamp2 or Antp was added. Fluorescence spectroscopic measurements further show that the tryptophan of the three peptides may interact with the hydrophobic region of the POPC/POPG bilayers, thus resulting in a change in the conformational order of the POPC/POPG acyl chains. This study may provide valuable insight into the dynamics and flexibility of lipid bilayers with different lipids and highlights the importance of aromatic residues in peptide binding to the membrane.

Aromatic residues play important roles in modulating the function of proteins and binding of proteins to biomembranes. However, the effect of small molecules which contain aromatic residues on the packing of unsaturated lipid acyl chains and the dynamics properties in membrane bilayers remains unclear. The membrane interactions of a series of amino acid methyl esters have been investigated to establish how different amino acids influence lipid acyl chains packing. Attenuated total reflectance-Fourier transform infrared spectroscopic (ATR-FTIR) measurements show that the CH and CC stretching vibrational bands of 2-oleoyl-1-pamlitoyl-sn-glyecro-3-phosphocholine (POPC) present evident red-shifts when aromatic amino acid methyl esters are mixed into membrane bilayers. Quantum mechanical (QM) calculations further demonstrate that the red shifts of the CH and CC stretching modes for POPC acyl chains are not through a direct interaction between the side chain groups and the HCCH motif but are mainly caused by the conformational change of POPC acyl chains, in which the HCCH motif is packed closed to the other one of an adjacent acyl chain with enhancement by dispersion interactions. The implication of these findings is that the packing of POPC acyl chains can be significantly influenced by aromatic amino acid methyl esters. This work may provide valuable insight into the effect on the lipid acyl chains packing by small molecules interacting with membranes and offer possibilities for the dynamic control of membrane fusion and binding of proteins to the membrane.