DOTAP: Structure, hydration, and the counterion effect.

The cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is one of the original synthetic cationic lipids used for the liposomal transfection of oligonucleotides in gene therapy. The key structural element of DOTAP is its quaternary ammonium headgroup that is responsible for interactions with both nucleic acids and target cell membranes. Because these interactions are fundamental to the design of a major class of transfection lipids, it is important to understand the structure of DOTAP and how it interacts with halide counterions. Here, we use x-ray and neutron diffraction techniques to examine the structure of DOTAP and how chloride (Cl-) and iodide (I-) counterions alter the hydration properties of the DOTAP headgroup. A problem of particular interest is the poor solubility of DOTAP/I- in water solutions. Our results show that the poor solubility results from very tight binding of the I- counterion to the headgroup and the consequent expulsion of water. The structural principles we report here are important for assessing the suitability of DOTAP and its quaternary ammonium derivatives for transfection.

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function.

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.

Computed Free Energies of Peptide Insertion into Bilayers are Independent of Computational Method.

We show that the free energy of inserting hydrophobic peptides into lipid bilayer membranes from surface-aligned to transmembrane inserted states can be reliably calculated using atomistic models. We use two entirely different computational methods: high temperature spontaneous peptide insertion calculations as well as umbrella sampling potential-of-mean-force (PMF) calculations, both yielding the same energetic profiles. The insertion free energies were calculated using two different protein and lipid force fields (OPLS protein/united-atom lipids and CHARMM36 protein/all-atom lipids) and found to be independent of the simulation parameters. In addition, the free energy of insertion is found to be independent of temperature for both force fields. However, we find major difference in the partitioning kinetics between OPLS and CHARMM36, likely due to the difference in roughness of the underlying free energy surfaces. Our results demonstrate not only a reliable method to calculate insertion free energies for peptides, but also represent a rare case where equilibrium simulations and PMF calculations can be directly compared.

Correction to: Computed Free Energies of Peptide Insertion into Bilayers are Independent of Computational Method.

The original version of the article unfortunately contained an error in NIH support grant number RO1-GM74639 in the Acknowledgements section. The correct grant number is RO1-GM74637. This has been corrected with this erratum.

Anomalous behavior of water inside the SecY translocon.

The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.

Interleaflet mixing and coupling in liquid-disordered phospholipid bilayers.

Organized as bilayers, phospholipids are the fundamental building blocks of cellular membranes and determine many of their biological functions. Interactions between the two leaflets of the bilayer (interleaflet coupling) have been implicated in the passage of information through membranes. However, physically, the meaning of interleaflet coupling is ill defined and lacks a structural basis. Using all-atom molecular dynamics simulations of fluid phospholipid bilayers of five different lipids with differing degrees of acyl-chain asymmetry, we have examined interleaflet mixing to gain insights into coupling. Reasoning that the transbilayer distribution of terminal methyl groups is an appropriate measure of interleaflet mixing, we calculated the transbilayer distributions of the acyl chain terminal methyl groups for five lipids: dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), stearoyloleoylphosphatidylcholine (SOPC), oleoylmyristoylphosphatidylcholine (OMPC), and dimyristoylphosphatidylcholine (DMPC). We observed in all cases very strong mixing across the bilayer midplane that diminished somewhat with increasing acyl-chain ordering defined by methylene order parameters. A hallmark of the interleaflet coupling idea is complementarity, which postulates that lipids with short alkyl chains in one leaflet will preferentially associate with lipids with long alkyl chains in the other leaflet. Our results suggest a much more complicated picture for thermally disordered bilayers that we call distributed complementarity, as measured by the difference in the peak positions of the sn-1 and sn-2 methyl distributions in the same leaflet.

Transmembrane helices containing a charged arginine are thermodynamically stable.

Hydrophobic amino acids are abundant in transmembrane (TM) helices of membrane proteins. Charged residues are sparse, apparently due to the unfavorable energetic cost of partitioning charges into nonpolar phases. Nevertheless, conserved arginine residues within TM helices regulate vital functions, such as ion channel voltage gating and integrin receptor inactivation. The energetic cost of arginine in various positions along hydrophobic helices has been controversial. Potential of mean force (PMF) calculations from atomistic molecular dynamics simulations predict very large energetic penalties, while in vitro experiments with Sec61 translocons indicate much smaller penalties, even for arginine in the center of hydrophobic TM helices. Resolution of this conflict has proved difficult, because the in vitro assay utilizes the complex Sec61 translocon, while the PMF calculations rely on the choice of simulation system and reaction coordinate. Here we present the results of computational and experimental studies that permit direct comparison with the Sec61 translocon results. We find that the Sec61 translocon mediates less efficient membrane insertion of Arg-containing TM helices compared with our computational and experimental bilayer-insertion results. In the simulations, a combination of arginine snorkeling, bilayer deformation, and peptide tilting is sufficient to lower the penalty of Arg insertion to an extent such that a hydrophobic TM helix with a central Arg residue readily inserts into a model membrane. Less favorable insertion by the translocon may be due to the decreased fluidity of the endoplasmic reticulum (ER) membrane compared with pure palmitoyloleoyl-phosphocholine (POPC). Nevertheless, our results provide an explanation for the differences between PMF- and experiment-based penalties for Arg burial.

Biophysical dissection of membrane proteins.

The first atomic-resolution structure of a membrane protein was solved in 1985. Twenty-four years and more than 180 unique structures later, what have we have learned? An examination of the atomic details of several diverse membrane proteins reveals some remarkable biophysical features and suggests that we can expect to achieve much more in the decades to come.

Structure and hydration of membranes embedded with voltage-sensing domains.

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.

Galactoside-binding site in LacY.

Although an X-ray crystal structure of lactose permease (LacY) has been presented with bound galactopyranoside, neither the sugar nor the residues ligating the sugar can be identified with precision at ~3.5 Å. Therefore, additional evidence is important for identifying side chains likely to be involved in binding. On the basis of a clue from site-directed alkylation suggesting that Asn272, Gly268, and Val264 on one face of helix VIII might participate in galactoside binding, molecular dynamics simulations were conducted initially. The simulations indicate that Asn272 (helix VIII) is sufficiently close to the galactopyranosyl ring of a docked lactose analogue to play an important role in binding, the backbone at Gly268 may be involved, and Val264 does not interact with the bound sugar. When the three side chains are subjected to site-directed mutagenesis, with the sole exception of mutant Asn272 → Gln, various other replacements for Asn272 either markedly decrease affinity for the substrate (i.e., high KD) or abolish binding altogether. However, mutant Gly268 → Ala exhibits a moderate 8-fold decrease in affinity, and binding by mutant Val264 → Ala is affected only minimally. Thus, Asn272 and possibly Gly268 may comprise additional components of the galactoside-binding site in LacY.

Charge composition features of model single-span membrane proteins that determine selection of YidC and SecYEG translocase pathways in Escherichia coli.

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum.

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature 427:36-44; Zimmer J, et al. (2008) Nature 455:936-943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182-17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol · Å(2)), the solvation parameter for transfer from translocon to bilayer is 6-10 cal/(mol · Å(2)), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.

Conformational states of melittin at a bilayer interface.

The distribution of peptide conformations in the membrane interface is central to partitioning energetics. Molecular-dynamics simulations enable characterization of in-membrane structural dynamics. Here, we describe melittin partitioning into dioleoylphosphatidylcholine lipids using CHARMM and OPLS force fields. Although the OPLS simulation failed to reproduce experimental results, the CHARMM simulation reported was consistent with experiments. The CHARMM simulation showed melittin to be represented by a narrow distribution of folding states in the membrane interface.

Hydrogen bond dynamics in membrane protein function.

Changes in inter-helical hydrogen bonding are associated with the conformational dynamics of membrane proteins. The function of the protein depends on the surrounding lipid membrane. Here we review through specific examples how dynamical hydrogen bonds can ensure an elegant and efficient mechanism of long-distance intra-protein and protein-lipid coupling, contributing to the stability of discrete protein conformational substates and to rapid propagation of structural perturbations. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Hydrogen-bond energetics drive helix formation in membrane interfaces.

The free energy cost ΔG of partitioning many unfolded peptides into membrane interfaces is unfavorable due to the cost of partitioning backbone peptide bonds. The partitioning cost is dramatically reduced if the peptide bonds participate in hydrogen bonds. The reduced cost underlies secondary structure formation by amphiphilic peptides partitioned into membrane interfaces through a process referred to as partitioning-folding coupling. This coupling is characterized by the free energy reduction per residue, ∆G(res) that drives folding. There is some debate about the correct value of ∆G(res) and its dependence on the hydrophobic moment (µ(H)) of amphiphilic α-helical peptides. We show how to compute ∆G(res) correctly. Using published data for two families of peptides with different hydrophobic moments and charges, we find that ∆G(res) does not depend upon µ(H). The best estimate of ∆G(res) is -0.37 ± 0.02 kcal mol(-1). This article is part of a Special Issue entitled: Membrane protein structure and function.

Water wires in atomistic models of the Hv1 proton channel.

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. This article is part of a Special Issue entitled: Membrane protein structure and function.

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel.

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.

Molecular code for transmembrane-helix recognition by the Sec61 translocon.

Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

Fifty Years of Biophysics at the Membrane Frontier.

The author first describes his childhood in the South and the ways in which it fostered the values he has espoused throughout his life, his development of a keen fascination with science, and the influences that supported his progress toward higher education. His experiences in ROTC as a student, followed by two years in the US Army during the Vietnam War, honed his leadership skills. The bulk of the autobiography is a chronological journey through his scientific career, beginning with arrival at the University of California, Irvine in 1972, with an emphasis on the postdoctoral students and colleagues who have contributed substantially to each phase of his lab's progress. White's fundamental findings played a key role in the development of membrane biophysics, helping establish it as fertile ground for research. A story gradually unfolds that reveals the deeply collaborative and painstakingly executed work necessary for a successful career in science.

Microscopic origin of gating current fluctuations in a potassium channel voltage sensor.

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-µs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD.