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1.  Allosteric mechanism of water channel gating by Ca2+–calmodulin 
Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.
doi:10.1038/nsmb.2630
PMCID: PMC3766450  PMID: 23893133
aquaporin (AQP); gating; calmodulin (CaM); electron microscopy (EM); molecular dynamics (MD); calcium regulation; water channel; membrane protein complex
2.  WATER WIRES IN ATOMISTIC MODELS OF THE HV1 PROTON CHANNEL 
Biochimica et Biophysica Acta  2011;1818(2):286-293.
The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions.
doi:10.1016/j.bbamem.2011.07.045
PMCID: PMC3245885  PMID: 21843503
Voltage-gated ion channels; Voltage-sensing domains; Membrane Proteins; Molecular Dynamics Simulations
3.  PROTON-COUPLED DYNAMICS IN LACTOSE PERMEASE 
Structure (London, England : 1993)  2012;20(11):1893-1904.
Summary
Lactose permease of Escherichia coli (LacY) catalyzes symport of a galactopyranoside and an H+ via an alternating access mechanism. The transition from an inward- to an outward-facing conformation of LacY involves sugar-release followed by deprotonation. Because the transition depends intimately upon the dynamics of LacY in a bilayer environment, molecular dynamics (MD) simulations may be the only means of following the accompanying structural changes in atomic detail. We describe here MD simulations of wild-type apo LacY in phosphatidylethanolamine (POPE) lipids that features two protonation states of the critical Glu325. While the protonated system displays configurational stability, deprotonation of Glu325 causes significant structural rearrangements that bring into proximity sidechains important for H+ translocation and sugar binding and closes the internal cavity. Moreover, protonated LacY in phosphatidylcholine (DMPC) lipids shows that the observed dynamics are lipid-dependent. Together, the simulations describe early dynamics of the inward-to-outward transition of LacY that agree well with experimental data.
doi:10.1016/j.str.2012.08.021
PMCID: PMC3496080  PMID: 23000385
lactose permease; protonation states; protein-lipid interactions; MD simulations
4.  Assembly and Stability of α-Helical Membrane Proteins 
Soft matter  2012;8(30):7742-7752.
Grease to grease – this is how one might begin to describe the tendency of hydrophobic stretches in protein amino acid sequences to form transmembrane domains. While this simple rule contains a lot of truth, the mechanisms of membrane protein folding, the insertion of hydrophobic protein domains into the lipid bilayer, and the apparent existence of highly polar residues in some proteins in the hydrophobic membrane core are subjects of lively debate – an indication that many details remain unresolved. Here, we present a historical survey of recent insights from experiments and computational studies into the rules and mechanisms of α-helical membrane protein assembly and stability.
doi:10.1039/C2SM25402F
PMCID: PMC3500387  PMID: 23166562
5.  Coupling between the Voltage-sensing and Pore Domains in a Voltage-gated Potassium Channel 
Biochimica et biophysica acta  2012;1818(7):10.1016/j.bbamem.2012.02.029.
Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.
doi:10.1016/j.bbamem.2012.02.029
PMCID: PMC3387523  PMID: 22425907
6.  A Transporter Converted into a Sensor, a Phototaxis Signaling Mutant of Bacteriorhodopsin at 3.0 Å 
Journal of molecular biology  2011;415(3):455-463.
Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 Å resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C14 group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174–Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.
doi:10.1016/j.jmb.2011.11.025
PMCID: PMC3628824  PMID: 22123198
microbial rhodopsins; sensory rhodopsin II; bacteriorhodopsin; light sensor; phototaxis
7.  Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers 
Biochimica et Biophysica Acta  2011;1818(2):126-134.
Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host-guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210 is well tolerated in OmpLA and that the DLPC bilayers accommodate this perturbation by creating a water dimple that allows the Arg side chain to remain hydrated. Lipid head groups visit the dimple and can hydrogen bond with Arg, but these interactions are transient. Overall, our study demonstrates the unique advantages of this molecular system because it can be interrogated by both computational and experimental practitioners, and it sets the stage for free energy calculations in a system for which there is unambiguous experimental data.
doi:10.1016/j.bbamem.2011.07.016
PMCID: PMC3233656  PMID: 21816133
Membrane proteins; protein stability; thermodynamics; computation; solvation; phospholipid bilayers
8.  Structural Dynamics Of The S4 Voltage-Sensor Helix In Lipid Bilayers Lacking Lipid Phosphates 
The journal of physical chemistry. B  2011;115(27):8732-8738.
Voltage-dependent K+ (Kv) channels require lipid phosphates for functioning. The S4 helix, which carries the gating charges in the voltage-sensing domain (VSD), inserts into membranes while being stabilized by a protein-lipid interface in which lipid phosphates play an essential role. To examine the physical basis of the protein-lipid interface in the absence of lipid phosphates, we performed molecular dynamics (MD) simulations of a KvAP S4 variant (S4mut) in bilayers with and without lipid phosphates. We find that in dioleoyltrimethylammoniumpropane (DOTAP) bilayers lacking lipid phosphates, the gating charges are solvated by anionic counterions and, hence, lack the bilayer support provided by phosphate-containing palmitoyloleoylglycerophosphocholine (POPC) bilayers. The result is a water-permeable bilayer with a significantly smaller deformations around the peptide. Together, these results provide an explanation for the non-functionality of VSDs in terms of a destabilizing protein-lipid interface.
doi:10.1021/jp2001964
PMCID: PMC3140535  PMID: 21692541
molecular dynamics simulations; dioleoyltrimethylammoniumpropane (DOTAP); voltage-dependent K+ (Kv) channels; protein-lipid interactions
9.  Dynamics of SecY Translocons with Translocation-Defective Mutations 
Summary
The SecY/Sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (Sec61) or the plasma membrane of prokaryotes (SecY), mediates the transmembrane secretion or insertion of nascent proteins. Mutations that permit the secretion of nascent proteins with defective signal sequences (Prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. The crystallographic structure of SecYEβ from Methanococcus jannaschii revealed widespread distribution of mutations causing topogenesis defects, but not their molecular mechanisms. Based upon prolonged molecular dynamics simulations of wild-type M. jannaschii SecYEβ and an extensive sequence-conservation analysis, we show that the closed-state of the translocon is stabilized by hydrogen-bonding interactions of numerous highly conserved amino acids. Perturbations induced by mutation at various locations are rapidly relayed to the plug segment that seals the wild-type closed-state translocon, leading to displacement and increased hydration of the plug.
doi:10.1016/j.str.2010.04.010
PMCID: PMC2909450  PMID: 20637421
protein biosynthesis; SecY/Sec61 translocon; hydrogen bonding; molecular dynamics simulations
10.  Update of the CHARMM all-atom additive force field for lipids: Validation on six lipid types 
The journal of physical chemistry. B  2010;114(23):7830-7843.
A significant modification to the additive all-atom CHARMM lipid force field (FF) is developed and applied to phospholipid bilayers with both choline and ethanolamine containing head groups and with both saturated and unsaturated aliphatic chains. Motivated by the current CHARMM lipid FF (C27 and C27r) systematically yielding values of the surface area per lipid that are smaller than experimental estimates and gel-like structures of bilayers well above the gel transition temperature, selected torsional, Lennard-Jones and partial atomic charge parameters were modified by targeting both quantum mechanical (QM) and experimental data. QM calculations ranging from high-level ab initio calculations on small molecules to semi-empirical QM studies on a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer in combination with experimental thermodynamic data were used as target data for parameter optimization. These changes were tested with simulations of pure bilayers at high hydration of the following six lipids: DPPC, 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE); simulations of a low hydration DOPC bilayer were also performed. Agreement with experimental surface area is on average within 2%, and the density profiles agree well with neutron and x-ray diffraction experiments. NMR deuterium order parameters (SCD) are well predicted with the new FF, including proper splitting of the SCD for the aliphatic carbon adjacent to the carbonyl for DPPC, POPE, and POPC bilayers. The area compressibility modulus and frequency dependence of 13C NMR relaxation rates of DPPC, and the water distribution of low hydration DOPC bilayers also agree well with experiment. Accordingly, the presented lipid FF, referred to as C36, allows for molecular dynamics simulations to be run in the tensionless ensemble (NPT), and is anticipated to be of utility for simulations of pure lipids systems as well as heterogeneous systems including membrane proteins.
doi:10.1021/jp101759q
PMCID: PMC2922408  PMID: 20496934
11.  Festschrift in the Honor of Stephen H. White’s 70th Birthday 
The Journal of Membrane Biology  2010;239(1-2):1-3.
The Symposium ‘Frontiers in membrane and membrane protein biophysics: experiments and theory’, held this year at the University of California, Irvine (August 19–20), celebrated the 70th Birthday of Stephen H. White by bringing together distinguished experimentalists and theoreticians to discuss the state of the art and future challenges in the field of membrane and membrane protein biophysics. The meeting and this special issue highlight the highly interdisciplinary nature of membrane and membrane protein biophysics, and the tremendous contributions that S. H. White and his lab have brought to the field.
doi:10.1007/s00232-010-9337-3
PMCID: PMC3030939  PMID: 21191785
S. H. White; Membrane and membrane protein biophysics
12.  Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments 
The Journal of Membrane Biology  2010;239(1-2):35-48.
Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.
doi:10.1007/s00232-010-9330-x
PMCID: PMC3030942  PMID: 21127848
Ion permeation mechanisms; Membrane transport-theoretical or experimental; Voltage-dependent ion channels; Membrane biophysics
13.  Sensitivity of 2D IR Spectra to Peptide Helicity: A Concerted Experimental and Simulation Study of an Octapeptide 
The journal of physical chemistry. B  2009;113(35):12037-12049.
We have investigated the sensitivity of two-dimensional infrared (2D IR) spectroscopy to peptide helicity with an experimental and theoretical study of Z-[L-(αMe)Val]8-OtBu in CDCl3. 2D IR experiments were carried out in the amide-I region under the parallel and the double-crossed polarization configurations. In the latter polarization configuration, the 2D spectra taken with the rephasing and nonrephasing pulse sequences exhibit a doublet feature and a single peak, respectively. These cross-peak patterns are highly sensitive to the underlying peptide structure. Spectral calculations were performed on the basis of a vibrational exciton model, with the local mode frequencies and couplings calculated from snapshots of molecular dynamics (MD) simulation trajectories using six different models for the Hamiltonian. Conformationally variant segments of the MD trajectory, while reproducing the main features of the experimental spectra, are characterized by extraneous features, suggesting that the structural ensembles sampled by the simulation are too broad. By imposing periodic restraints on the peptide dihedral angles with the crystal structure as a reference, much better agreement between the measured and the calculated spectra was achieved. The result indicates that the structure of Z-[L-(αMe)Val]8-OtBu in CDCl3 is a fully developed 310-helix with only a small fraction of α-helical or nonhelical conformations in the middle of the peptide. Of the four different combinations of pulse sequences and polarization configurations, the nonrephasing double-crossed polarization 2D IR spectrum exhibits the highest sensitivity in detecting conformational variation. Of the six local mode frequency models tested, the electrostatic maps of Mukamel and Cho perform the best. Our results show that the high sensitivity of 2D IR spectroscopy can provide a useful basis for developing methods to improve the sampling accuracy of force fields and for characterizing the relative merits of the different protocols for the Hamiltonian calculation.
doi:10.1021/jp901504r
PMCID: PMC2932706  PMID: 19496555
14.  Direct comparisons of experimental and calculated neutron structure factors of pure solvents as a method for force field validation 
The journal of physical chemistry. B  2007;111(45):12941-12944.
In the present letter we directly compare neutron structure factors calculated from force field (FF)-based molecular dynamics simulations with experimental structure factors for water, methanol, and tetrahydrofuran (THF). For water, the difference in measured structure factors is more significant than differences between the FFs. It is shown that the inclusion of electronic polarization in the force field improves the agreement with experiment for the more polar methanol while the results are comparable for the additive and polarizable FF models of the less polar THF. The data presented here confirm that comparing calculated scattering profiles from FF based MD simulations to measured neutron structure factors is a promising method for FF validation and development.
doi:10.1021/jp076501p
PMCID: PMC2568990  PMID: 17958354

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