Pulling single bacteriorhodopsin out of a membrane: Comparison of simulation and experiment

Mechanical unfolding of single bacteriorhodopsins from a membrane bilayer is studied using molecular dynamics simulations. The initial conformation of the lipid membrane is determined through all-atom simulations and then its coarse-grained representation is used in the studies of stretching. A Go-like model with a realistic contact map and with Lennard-Jones contact interactions is applied to model the protein-membrane system. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on bacteriorhodopsin at different temperatures and predicts a lack of symmetry in the choice of the terminus to pull by. It also illustrates the decisive role of the interactions of the protein with the membrane in determining the force pattern and thus the stability of transmembrane proteins.     

Refolding of a membrane protein in a microfluidics reactor

Membrane protein production for structural studies is often hindered by the formation of non-specific aggregates from which the protein has to be denatured and then refolded to a functional state. We developed a new approach, which uses microfluidics channels, to refold protein correctly in quantities sufficient for structural studies. Green fluorescent protein (GFP), a soluble protein, and bacteriorhodopsin (BR), a transmembrane protein, were used to demonstrate the efficiency of the process. Urea-denatured GFP refolded as the urea diffused away from the protein, forming in the channel a uniform fluorescent band when observed by confocal microscopy. Sodium dodecyl sulphate-denatured BR refolded within the channel on mixing with detergent–lipid mixed micelles. The refolding, monitored by absorbance spectroscopy, was found to be flow rate dependent. This potential of microfluidic reactors for screening protein-folding conditions and producing protein would be particularly amenable for high-throughput applications required in structural genomics. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.     

Folding and stretching in a Go-like model of titin

Mechanical stretching of the I27 domain of titin and of its double and triple repeats are studied through molecular dynamics simulations of a Go-like model with Lennard-Jones contact interactions. We provide a thorough characterization of the system and correlate the sequencing of the folding and unraveling events with each other and with the contact order. The roles of cantilever stiffness and pulling rate are studied. Unraveling of tandem titin structures has a serial nature. The force-displacement curves in this coarse-grained model are similar to those obtained through all atom calculations.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Energetics and stability of transmembrane helix packing: a replica-exchange simulation with a knowledge-based membrane potential

The energetics and stability of the packing of transmembrane helices were investigated by Monte Carlo simulations with the replica-exchange method. The helices were modeled with a united atom representation, and the CHARMM19 force field was employed. Based on known experimental structures of membrane proteins, an implicit knowledge-based potential was developed to describe the helix-membrane interactions at the residue level, whose validity was tested through prediction of the orientations when single helices were inserted into a membrane. Two systems were studied in this article, namely the glycophorin A dimer, and helices A and B of Bacteriorhodopsin. For the glycophorin A dimer, the most stable structure (0.5 A away from the experimental structure) is mainly stabilized by the favorable helix-helix interactions, and has the most population regardless of the helix-membrane interaction. However, for helices A and B of Bacteriorhodopsin, it was found that the packing determined by helix-helix interactions is nonspecific, and a native-like structure (0.2 A from the experimental one) can be identified from several structural analogs as the most stable one only after applying the membrane potential. Our results suggest that the contribution from the helix-membrane interaction could be critical in the correct packing of transmembrane helices in the membrane.     

Interaction between dihydropyridines and phospholipid bilayers: a molecular dynamics simulation

Interaction of the calcium-channel antagonist dihydropyridines (DHPs), lacidipine and nifedipine, with a phospholipid bilayer was studied using 600 ps molecular dynamic simulations. We have constructed a double layer membrane model composed of 42 dimirystoyl-phosphatidylcholine molecules. The DHP molecules locate at about 7 Å from the centre of the membrane, inducing an asymmetry in the bilayer. While lacidipine did not induce significant local perturbations as judged by the gauche-trans isomerisation rate, nifedipine significantly decreased this rate, probably by producing a local rigidity of the membrane in the vicinity of the DHP.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Peptide building blocks from bacteriorhodopsin: Isolation and physicochemical characterization of two individual transmembrane segments

For protein engineering purposes, transmembrane segments of the structurally stable protein bacteriorhodopsin have been isolated and chemically characterized. Bacteriorhodopsin was cleaved by protease V8 fromStaphylococcus aureus to two fragments, V-1 and V-2. The V-2 fragment was separated by gel filtration in organic solvents and purified by reversed-phase FPLC. The fragment has been identified as the C-terminal, partially truncated double-loop of bacteriorhodopsin, including amino acids Val-167-Glu-232/4. Cleavage of V-2 by cyanogen bromide at the single Met-209 yielded two subfragments, which were purified to homogeneity by FPLC procedures. The N-terminal subfragment , consisted of a single transmembrane segment (helix F) of bacteriorhodopsin (Val-167-Met(Hse)-209). The C-terminal amphipathic subfragment , (Val-210-Glu-232/4) was identified as part of the C-terminal seventh helix of bacteriorhodopsin. Secondary structures of V-2, , and were investigated in organic solvents and micellar solutions. Native helical structures were partially retained in the solvent systems mentioned.     

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96 Asn; PM 374: Asp96 Gly; PM 384: Asp85 Glu) were grown in a synthetic medium containing (4-¹³C)-Asp. The mutant bacteriorhodopsins labeled with (4-¹³C)-Asp (37%–45%), and owing to the metabolism of Halobacteria also with (11-¹³C)-Trp (50%–100%), were isolated as purple membranes and ¹³C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989 a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85 Glu) has an absorption maximum at 610 run. It can be converted into a purple form (_max = 5.40 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.Abbreviations CHAPSO 3-(3-cholamidopropyl)-dimethylammonio2-hydroxy-1-propane sulfonate - CP crosspolarization - bR bacteriorhodopsin - FTIR Fourier-transform-infrared - FID free induction decay - IR Infrared - MASS Magic angle sample spinning - NMR Nuclear magnetic resonance - TMS tetramethylsilane This research was supported by the Deutsche Forschungsgemeinschaft #SFB 60G9 Offprint requests to: M. Engelhard     

A multidomain outer membrane protein from Pasteurella multocida: Modelling and simulation studies of PmOmpA

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane β-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane β-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the β-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane β-barrels, opening the possibility of in silico proteomics approaches to such proteins.     

Fusion of bacteriorhodopsin with submitochondrial particles yields a new system with retention of energy coupling and acquisition of photophosphorylation activity

Submitochondrial particles were fused with purple membranes of Halobacterium halobium cells by means of a freeze-thawe sonication procedure. It is reported that fusion of inner mitochondrial membranes with a bacterial membrane yields a new particle which shows not only retention of redox- and photon-linked energy-coupling activities, but also creation of an additional energy-coupling process, light-driven ATP synthesis.     

A prokaryotic glutamate receptor: homology modelling and molecular dynamics simulations of GluR0

GluR0 is a prokaryotic homologue of mammalian glutamate receptors that forms glutamate-activated, potassium-selective ion channels. The topology of its transmembrane (TM) domain is similar to that of simple potassium channels such as KcsA. Two plausible alignments of the sequence of the TM domain of GluR0 with KcsA are possible, differing in the region of the P helix. We have constructed homology models based on both alignments and evaluated them using 6 ns duration molecular dynamics simulations in a membrane-mimetic environment. One model, in which an insertion in GluR0 relative to KcsA is located in the loop between the M1 and P helices, is preferred on the basis of lower structural drift and maintenance of the P helix conformation during simulation. This model also exhibits inter-subunit salt bridges that help to stabilise the TM domain tetramer. During the simulation, concerted K(+) ion-water movement along the selectivity filter is observed, as is the case in simulations of KcsA. K(+) ion exit from the central cavity is associated with opening of the hydrophobic gate formed by the C-termini of the M2 helices. In the intact receptor the opening of this gate will be controlled by interactions with the extramembranous ligand-binding domains.     

Carnitine palmitoyltransferase 2: analysis of membrane association and complex structure with a substrate analog

The mitochondrial membrane-associated carnitine palmitoyltransferase system is a validated target for the treatment of type 2 diabetes mellitus. To further facilitate structure-based drug discovery, we determined the crystal structure of rat CPT-2 (rCPT-2) in complex with the substrate analogue palmitoyl-aminocarnitine at 1.8A resolution. Biochemical analyses revealed a strong effect of this compound on rCPT-2 activity and stability. Using a computational approach we examined the membrane association of rCPT-2. The protein interacts with the membrane as a functional monomer and the calculations confirm the presence of a membrane association domain that consists of layers of hydrophobic and positively charged residues.     

Interactions of oritavancin, a new lipoglycopeptide derived from vancomycin, with phospholipid bilayers: Effect on membrane permeability and nanoscale lipid membrane organization

Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (< 5 min) and complete (> 95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0-600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 μM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin.     

Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor

The recent crystal structures of the voltage-gated potassium channel KvAP and its isolated voltage-sensing 'paddle' (composed of segments S1-S4) challenge existing models of voltage gating and raise a number of questions about the structure of the physiologically relevant state. We investigate a possible gating mechanism based on the crystal structures in a 10 ns steered molecular dynamics simulation of KvAP in a membrane-mimetic octane layer. The structure of the full KvAP protein has been modified by restraining the S2-S4 domain to the conformation of the isolated high-resolution paddle structure. After an initial relaxation, the paddle tips are pulled through the membrane from the intracellular to the extracellular side, corresponding to a putative change from closed to open. We describe the effect of this large-scale motion on the central pore domain, which remains largely unchanged, on the protein hydrogen-bonding network and on solvent. We analyze the motion of the S3b-S4 portion of the protein and propose a possible coupling mechanism between the paddle motion and the opening of the channel. Interactions between the arginine residues in S4, solvent and chloride ions are likely to play a role in the gating charge.     

Progress in the analysis of membrane protein structure and function

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.     

Post-testicular development of a novel membrane substructure within the equatorial segment of ram,bull, boar,and goat spermatozoa as viewed by atomic force microscopy

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca(2+) ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.