Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state 2H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp2656.48 residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

Visual rhodopsin is an important archetype for G‐protein‐coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent‐swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force‐based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin‐II state. Moreover, we discovered that binding and release of the C‐terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration–dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G‐protein cycle.

Molecular dynamics (MD) simulations were used to characterize the equilibrium size, shape, hydration, and self-assembly of dodecylphosphocholine (DPC) and dodecyl-β-D-maltoside (DDM) micelles. We show that DPC molecules self-assemble to form micelles with sizes within the range reported in the experimental literature. The equilibrium shape of DPC and DDM micelles as well as associated micellar radii are in agreement with small-angle X-ray scattering (SAXS) experiments and theoretical packing parameters. In addition, we show that hydration of the micelle interior is limited; however, flexibility of the acyl chains leads to dynamic encounters with the solvated outer shell of the micelle, providing an explanation for long-standing differences in models of micelle hydration. Altogether, our results provide fundamental understanding of physical characteristics of micelles that can be utilized to study other types of detergents and proteomicelle complexes.

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Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)—the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state 2H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states.

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.

The pH-low insertion peptide (pHLIP) is used for targeted delivery of drug cargoes to acidic tissues such as tumors. The extracellular acidosis found in solid tumors triggers pHLIP to transition from a membrane-adsorbed state to fold into a transmembrane a-helix. Different factors influence the acidity required for pHLIP to insert into lipid membranes. One of them is the lipid headgroup composition, which defines the electrostatic profile of the membrane. However, the molecular interactions that drive the adsorption of pHLIP to the bilayer surface are poorly understood. In this study, we combine biophysical experiments and all-atom molecular dynamics simulations to understand the role played by electrostatics in the interaction between pHLIP and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. We observed that the solution ionic strength affects the structure of pHLIP at the membrane surface as well as the acidity needed for different steps in the membrane insertion process. In particular, our simulations revealed that an increase in ionic strength affected both pHLIP and the bilayer; the coordination of sodium ions with the C-terminus of pHLIP led to localized changes in helicity, whereas the coordination of sodium ions with the phosphate moiety of the phosphocholine headgroups had a condensing effect on our model bilayer. These results are relevant to our understanding of environmental influences on the ability of pHLIP to adsorb to the cell membrane and are useful in our fundamental understanding of the absorption of pH-responsive peptides and cell-penetrating peptides.

Proteorhodopsin (PR) is a light-driven proton pump that is most notable for ushering in the discovery of an ever-increasing number of microbial retinal proteins that are at the forefront of fields such as optogenetics. Two variants, blue (BPR) and green (GPR) proteorhodopsin, have evolved to harvest light at different depths of the ocean. The color-tuning mechanism in PR is controlled by a single residue at position 105: in BPR it is a glutamine, whereas in GPR it is a leucine. Although the majority of studies on the spectral tuning mechanism in PR have focused on GPR, detailed understanding of the electronic environment responsible for spectral tuning in BPR is lacking. In this work, several BPR models were investigated using quantum mechanics/molecular mechanics (QM/MM) calculations to obtain fundamental insights into the color tuning mechanism of BPR. We find that the molecular mechanism of spectral tuning in BPR depends on two geometric parameters, the bond length alternation and the torsion angle deviation of the all-trans-retinyl chromophore. Both parameters are influenced by the strength of the hydrogen-bonded networks in the chromophore-binding pocket, which shows how BPR is different from other microbial rhodopsins.