In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

Efficient molecular mechanics simulations of the folding,orientation, and assembly of peptides in lipid bilayers using an implicit atomic solvation model

Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host–guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.     

Two‐Dimensional Crowding Uncovers a Hidden Conformation of α‐Synuclein

The intrinsically disordered protein (IDP), α‐synuclein (αS), is well‐known for phospholipid membrane binding‐coupled folding into tunable helical conformers. Here, using single‐molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction–folding landscape of αS can be tuned by two‐dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS–membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher‐affinity module remains membrane‐bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane‐binding IDPs with multiple binding modes.     

Insertion and assembly of membrane proteins via simulation

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular dynamics simulations have been used to reveal the mechanisms of self-assembly of protein/membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely, glycophorin (a simple alpha-helical bundle) and OmpA (a beta-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer self-assembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest that a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.     

Direct simulation of early-stage sec-facilitated protein translocation

Direct simulations reveal key mechanistic features of early-stage protein translocation and membrane integration via the Sec-translocon channel. We present a novel computational protocol that combines non-equilibrium growth of the nascent protein with microsecond timescale molecular dynamics trajectories. Analysis of multiple, long timescale simulations elucidates molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon lateral gate (LG), large lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrate the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups are shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (N(cyt)/C(exo)) integral membrane protein topology, and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer are shown to stabilize configurations that are consistent with the Type III (N(exo)/C(cyt)) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence.     

Real-Time Observation of Protein Transport across Membranes by Femtosecond Sum Frequency Generation Vibrational Spectroscopy?

Characterization of conformation kinetics of proteins at the interfaces is crucial for understanding the biomolecular functions and the mechanisms of interfacial biological action. But it requires to capture the dynamic structures of proteins at the interfaces with sufficient structural and temporal resolutions. Here, we demonstrate that a femtosecond sum frequency generation vibrational spectroscopy (SFG-VS) system developed by our group provides a powerful tool for monitoring the real-time peptide transport across the membranes with time resolution of less than one second. By probing the real-time SFG signals in the amide Ⅰ and amide A bands as WALP23 interacts with DMPG lipid bilayer, it is found that WALP23 is initially absorbed at the gel-phase DMPG bilayer with a random coil structure. The absorption of WALP23 on the surface leads to the surface charge reversal and thus changes the orientation of membrane-bound water. As the DMPG bilayer changes from gel phase into fluid phase, WALP23 inserts into the fluid-phase bilayer with its N-terminal end moving across the membrane, which causes the membrane dehydration and the transition of WALP23 conformation from random coil to mixed helix/loop structure and then to pure α-helical structure. The established system is ready to be employed in characterizing other interfacial fast processes, which will be certainly helpful for providing a clear physical picture of the interfacial phenomena.     

Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.     

Controlled delivery of proteins into bilayer lipid membranes on chip

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.     

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well‐defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell‐free expression systems, it has attracted attention as a next‐generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non‐volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell‐free expression systems as a drug screening system for future personalized medicine.     

Integrated prediction of protein folding and unfolding rates from only size and structural class

Protein stability, folding and unfolding rates are all determined by the multidimensional folding free energy surface, which in turn is dictated by factors such as size, structure, and amino-acid sequence. Work over the last 15 years has highlighted the role of size and 3D structure in determining folding rates, resulting in many procedures for their prediction. In contrast, unfolding rates are thought to depend on sequence specifics and be much more difficult to predict. Here we introduce a minimalist physics-based model that computes one-dimensional folding free energy surfaces using the number of aminoacids (N) and the structural class (α-helical, all-β, or α-β) as only protein-specific input. In this model N sets the overall cost in conformational entropy and the net stabilization energy, whereas the structural class defines the partitioning of the stabilization energy between local and non-local interactions. To test its predictive power, we calibrated the model empirically and implemented it into an algorithm for the PREdiction of Folding and Unfolding Rates (PREFUR). We found that PREFUR predicts the absolute folding and unfolding rates of an experimental database of 52 proteins with accuracies of ±0.7 and ±1.4 orders of magnitude, respectively (relative to experimental spans of 6 and 8 orders of magnitude). Such prediction uncertainty for proteins vastly varying in size and structure is only two-fold larger than the differences in folding (±0.34) and unfolding rates (±0.7) caused by single-point mutations. Moreover, PREFUR predicts protein stability with an accuracy of ±6.3 kJ mol(-1), relative to the 5 kJ mol(-1) average perturbation induced by single-point mutations. The remarkable performance of our simplistic model demonstrates that size and structural class are the major determinants of the folding landscapes of natural proteins, whereas sequence variability only provides the final 10-20% tuning. PREFUR is thus a powerful bioinformatic tool for the prediction of folding properties and analysis of experimental data.     

The balance between side-chain and backbone-driven association in folding of the α-helical influenza A transmembrane peptide

The correct balance between attractive, repulsive and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors, we sought a comparison of the folding between two 25-residues peptides, the influenza A M2 protein transmembrane domain (M2TM) and the 25-Ala (Ala₂₅). M2TM forms a stable α-helix as is shown by circular dichroism (CD) experiments. Molecular dynamics (MD) simulations with adaptive tempering show that M2TM monomer is more dynamic in nature and quickly interconverts between an ensemble of various α-helical structures, and less frequently turns and coils, compared to one α-helix for Ala₂₅. DFT calculations suggest that folding from the extended structure to the α-helical structure is favored for M2TM compared with Ala₂₅. This is due to CH⋯O attractive interactions which favor folding to the M2TM α-helix, and cannot be described accurately with a force field. Using natural bond orbital (NBO) analysis and quantum theory atoms in molecules (QTAIM) calculations, 26 CH⋯O interactions and 22 NH⋯O hydrogen bonds are calculated for M2TM. The calculations show that CH⋯O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total hydrogen bonding energy, when compared to NH⋯O, to the stabilization of the α-helix in M2TM. Further, a strengthening of NH⋯O hydrogen bonding interactions is calculated for M2TM compared to Ala₂₅. Additionally, these weak CH⋯O interactions can dissociate and associate easily leading to the ensemble of folded structures for M2TM observed in folding MD simulations.     

The native ensemble and folding of a protein molten-globule: functional consequence of downhill folding

The continually emerging functional significance of intrinsic disorder and conformational flexibility in proteins has challenged the long-standing dogma of a well-defined structure contributing to a specific function. Molten-globular states, a class of proteins with significant secondary-structure but a fluid hydrophobic core, is one such example. They have however been difficult to characterize due to the complexity of experimental data and lack of computational avenues. Here, we dissect the folding mechanism of the α-helical molten-globular protein NCBD from three fundamentally different approaches: statistical-mechanical variable barrier model, C(α)-based Gō-model and explicit water all-atom molecular dynamics (MD) simulations. We find that NCBD displays the characteristics of a one-state globally downhill folder but is significantly destabilized. Using simulation techniques, we generate a highly constrained but a heterogeneous native ensemble of the molten-globule for the first time that is consistent with experimental data including small angle X-ray scattering (SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR). The resulting native ensemble populates conformations reported in other bound-forms providing direct evidence to the mechanism of conformational selection for binding multiple partners in this domain. Importantly, our simulations reveal a connection between downhill folding and large conformational flexibility in this domain that has been evolutionarily selected and functionally exploited resulting in large binding promiscuity. Finally, the multimodel approach we employ here serves as a powerful methodology to study mechanisms and suggests that the thermodynamic features of molten-globules fall within the array of folding mechanisms available to small single-domain proteins.     

Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers

Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.     

Exploring and characterizing the folding processes of Lys- and Arg-containing Ala-based peptides: a molecular dynamics study

In this study, molecular dynamics simulations were carried out on Lys- and Arg-containing Ala-based peptides (i.e. Ace-(AAAAK)(n)A-NH(2) and Ace-(AAAAR)(n)A-NH(2), where n=1-4), in order to explore and characterize their folding processes. For the oligopeptides, the evolution of α-helical structure with regard to the whole conformation, as well as to each residue was investigated, and the helix-forming propensities were characterized. On the basis of the helicity curves, representing the alteration of average helicity as a function of time, the typical time values describing the folding processes and subprocesses were identified. In the case of each peptide, the evolution and role of helix-stabilizing, non-local and side-chain-to-backbone H-bonds were examined. The appearing i←i+4 H-bonds pointed out the role of these interactions in the stabilization of α-helical conformations, while the occurring i←i+3 H-bonds indicated the presence of β-turn or 3(10)-helical structures. Studying the formation and role of non-local and side-chain-to-backbone H-bonds led to the observation that these types of interactions produced an effect on the evolution of helical conformations, as well as on the folding processes.     

Microtechnologies for membrane protein studies

Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research.     

Detection of single ion channel activity on a chip using tethered bilayer membranes

Membrane-bound ion channels are promising biological receptors since they allow for the stochastic detection of analytes at high sensitivity. For stochastic sensing, it is necessary to measure the ion currents associated with single ion channel opening and closing events. However, this calls for stability, high reproducibility, and long lifetimes. A critical issue to overcome is the low stability of the ion channel environment, that is, the bilayer membrane. A promising technique to surmount this is to connect the lower part of the membrane to a surface forming a tethered bilayer membrane. By reconstituting the synthetic ion channel, gramicidin A, into a tethered bilayer as part of a microchip design, we have been able to record the activity of single ion channels. The observed activity was compared with that obtained by a conventional electrophysiology method, tip dipping, to confirm its authenticity. These findings allow for the construction of stable biosensors based on ion channels and provide a novel technique for the characterization of ion channel activity.     

Structural Investigation of Hybrid Peptide Foldamers Composed of α-Dipeptide Equivalent β-Oxy-δ5-amino Acids

Due to their equivalent lengths, δ-amino acids can serve as surrogates of α-dipeptides. However, δ-amino acids with proteinogenic side chains have not been well studied because of synthetic difficulties and because of their insolubility in organic solvents. Recently we reported the spontaneous supramolecular gelation of δ-peptides composed of β(O)-δ⁵-amino acids. Here, we report the incorporation of β(O)-δ⁵-amino acids as guests into the host α-helix, α,γ-hybrid peptide 12-helix and their single-crystal conformations. In addition, we studied the solution conformations of hybrid peptides composed of 1:1 alternating α and β(O)-δ⁵-amino acids. In contrast to the control α-helix structures, the crystal structure of peptides with β(O)-δ⁵-amino acids exhibit α-helical conformations consisting of both 13- and 10-membered H-bonds. The α,δ-hybrid peptide adopted mixed 13/11-helix conformation in solution with alternating H-bond directionality. Crystal-structure analysis revealed that the α,γ⁴-hybrid peptide accommodated the guest β(O)-δ⁵-amino acid without significant deviation to the overall helix folding. The results reported here emphasize that β(O)-δ⁵-amino acids with proteinogenic side chains can be accommodated into regular α-helix or 12-helix as guests without much deviation of the overall helix folding of the peptides.     

The d'--d--d' vertical triad is less discriminating than the a'--a--a' vertical triad in the antiparallel coiled-coil dimer motif

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.     

左苯丙胺在多巴胺第三受体分子通道中传输分子动力学模拟

左旋苯丙胺(又称左苯丙胺, RAT)在临床上被用于治疗多种病症,作用在中枢神经细胞多巴胺受体上,同时它具有依赖性和成瘾性。为了探讨RAT被用作药物的药理和成瘾机制,本文用分子模拟获得RAT与多巴胺第三受体(D₃R)复合蛋白优化结构,并且采用伞形样本平均力势(PMF)方法和卵磷脂脂质分子模拟生物膜,采用分子动力学模拟获得RAT在D₃R结构中分子通道运动轨迹和自由能变化。RAT通过D₃R结构中的功能分子通道,朝细胞外方向传输运动的自由能变化为91.4 kJ·mol^-1。RAT通过D₃R结构中的保护分子通道,朝细胞双层膜方向传输运动的自由能变化为117.7 kJ·mol^-1。自由能数值表明RAT分子更容易通过D₃R结构中的功能分子通道,发挥其功能作用,增大功能多巴胺分子的释放,导致包括依赖性和成瘾性多种功能效果。研究结果证明RAT被用作药物的药理和成瘾机制与它在多巴胺受体中的分子通道上传输动力学和机制有密切关联。