Helical Membrane Proteins Research Articles

Magic Angle Spinning and Oriented Sample Solid-State NMR Structural Restraints Combine for Influenza A M2 Protein Functional Insights

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.

A successful change of circumstance: a transition state for membrane protein folding

Knowledge of the transition state is key to understanding a reaction mechanism. This vital information has been lacking for integral membrane protein folding, but now recent advances have given insight into the structure of their folding transition state. This progress has arisen through the successful translation of a classical protein engineering method, ϕ value analysis, from water-soluble proteins to the hydrophobic, membrane-embedded protein class. This review covers the transition state for the folding of α helical membrane proteins. Helix formation in the transition state correlates with sequence position and the order of transmembrane insertion into the cell membrane, showing that in vitro measurements, in entirely different conditions to natural membranes, may reflect the cellular situation.

Developing a high-quality scoring function for membrane protein structures based on specific inter-residue interactions

Membrane proteins are of particular biological and pharmaceutical importance, and computational modeling and structure prediction approaches play an important role in studies of membrane proteins. Developing an accurate model quality assessment program is of significance to the structure prediction of membrane proteins. Few such programs are proposed that can be applied to a broad range of membrane protein classes and perform with high accuracy. We developed a new model scoring function Interaction-based Quality assessment (IQ), based on the analysis of four types of inter-residue interactions within the transmembrane domains of helical membrane proteins. This function was tested using three high-quality model sets: all 206 models of GPCR Dock 2008, all 284 models of GPCR Dock 2010, and all 92 helical membrane protein models of the HOMEP set. For all three sets, the scoring function can select the native structures among all of the models with the success rates of 93, 85, and 100% respectively. For comparison, these three model sets were also adopted for a recently published model assessment program for membrane protein structures, ProQM, which gave the success rates of 85, 79, and 92% separately. These results suggested that IQ outperforms ProQM when only the transmembrane regions of the models are considered. This scoring function should be useful for the computational modeling of membrane proteins.

Preferential Location of Amino Acids Across the Membrane in α-Helices and β-Barrel Membrane Protein Structures

Membrane proteins perform important physiological functions since they act as sensors for extra-cellular stimuli and gates for transport and communication. However, the basic forces and principles that drive membrane protein folding and assembly remain elusive. The key to the thermodynamic stability of membrane proteins is the complex physiochemical environment of the lipid bilayer. At the most basic level the hydrophobic core of the bilayer has been used to develop hydrophobicity scales that can identify putative transmembrane helices in membrane proteins. Recent refinements have utilized the realization that different amino acid types have strong preferences for particular insertion depths along the membrane normal, allowing a more detailed prediction and differentiation of buried, interfacial, and loop segments.However, prediction of beta-barrel membrane protein segments has so far resisted this analysis. This was primarily due to a lack of sufficient high resolution structures. Here we extend a method previously developed and successfully applied to alpha-helical membrane proteins to beta-barrels. We report the distribution of amino acids along the membrane normal for beta-barrels and compare the differences to alpha-helical proteins. For both protein types membrane interfaces are dominated by small residues such as glycine, alanine, and serine. Charged residues displayed non-symmetric distributions with charged residues generally preferring the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Even though there are many similarities the overall distributions are remarkably different between alpha and beta proteins, with beta-barrels displaying a much narrower hydrophobic core. We confirm that hydrophobic residues are the main driving force behind membrane protein insertion, while polar, charged and aromatic residues were found to be important for the correct orientation of the helix inside the membrane.

Association-Dissociation Dynamics of Transmembrane Helices as Detected by Single-Molecule Fluorescence Microscopy

Conformational fluctuations of helical membrane proteins among active-inactive conformations are crucial for their functions. Not only the amino acid sequence of the protein but also the composition of surrounding lipids should significantly affect the fluctuation, although the fundamental principles are mostly unknown. Experimental systems using model transmembrane helices and lipid bilayers have been proposed to be useful for direct measurements of elementary processes that determine the folding and conformational change of membrane proteins, such as insertion of the helix into lipid bilayers and helix-helix interaction in the bilayers. Here we show that real-time detection of self-association and dissociation of the transmembrane helix (AALALAA)3 is possible by single molecule FRET detection using the Cy3B- and Cy5- labeled helices incorporated into large unilamellar vesicles (LUVs, diameter ∼100 nm) composed of POPC. The LUVs were fixed on a glass surface via biotin-avidin interaction and observed under a total internal reflection fluorescence microscope. After simultaneous observation of Cy3B and Cy5 fluorescence with 10-20-ms time resolution, LUVs that had incorporated only one Cy3B- and one Cy5- labeled helices were selected based on the number of photobleaching steps, and single-molecular FRET from Cy3B to Cy5 was analyzed. FRET time course revealed that the helices fluctuate between monomer and dimer with a time scale of subseconds in the presence of cholesterol in the LUVs. The above experimental system will be useful for measurement of kinetics of helix-helix interactions in membranes with various lipid compositions.

Pattern of Amino Acid Substitutions in Transmembrane Domains of β-Barrel Membrane Proteins for Detecting Remote Homologs in Bacteria and Mitochondria

-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to -helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial -barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for -barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of -barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect -barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2–3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of -barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of -barrel membrane proteins, and will aid in the design of mutagenesis studies.

Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen

In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 Å. The transmembrane helix was found to have an average immersion depth of 5.4 Å, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.

Thermodynamics of Insertion and Self-Association of a Transmembrane Helix: A Lipophobic Interaction by Phosphatidylethanolamine

Thermodynamic parameters for the insertion and self-association of transmembrane helices are important for understanding the folding of helical membrane proteins. The lipid composition of bilayers would significantly affect these fundamental processes, although how is not well understood. Experimental systems using model transmembrane helices and lipid bilayers are useful for measuring and interpreting thermodynamic parameters (ΔG, ΔH, ΔS, and ΔC(p)) for the processes. In this study, the effect of the charge, phase, acyl chain unsaturation, and lateral pressure profile of bilayers on the membrane partitioning of the transmembrane helix (AALALAA)(3) was examined. Furthermore, the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) on the thermodynamics for insertion and self-association of the helix in host membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was investigated in detail. Interbilayer transfer of the helix monomer from POPC to POPC/POPE (1/1) bilayers was unfavorable (ΔG = +4.5 ± 2.9 kJ mol(-1) at 35 °C) due to an increase in enthalpy (ΔH = +31.1 ± 2.1 kJ mol(-1)). On the other hand, antiparallel dimerization of the helices in POPC/POPE (1/1) bilayers was enhanced compared with that in POPC bilayers (ΔΔG = -4.9 ± 0.2 kJ mol(-1) at 35 °C) due to a decrease in enthalpy (ΔΔH = -33.2 ± 1.5 kJ mol(-1)). A greater thickness of POPC/POPE bilayers only partially explained the observed effects. The residual effects could be related to changes in other physical properties such as higher lateral pressure in the hydrocarbon core in the PE-containing membrane. The origin of the enthalpy-driven "lipophobic" force that modulates the insertion and association of transmembrane helices will be discussed.

SOMPNN: an efficient non-parametric model for predicting transmembrane helices

Accurately predicting the transmembrane helices (TMH) in a helical membrane protein is an important but challenging task. Recent researches have demonstrated that statistics-based methods are promising routes to improve the TMH prediction accuracy. However, most of existing TMH predictors are parametric models and they have to make assumptions of several or even hundreds of adjustable parameters based on the underlying probability distribution, which is difficult when no a priori knowledge is available. Besides the performances of these parametric predictors significantly depend on the estimated parameters, some of them need to exploit the entire training dataset in the prediction stage, which will lead to low prediction efficiency and this problem will become even worse when dealing with large-scale dataset. In this paper, we propose a novel SOMPNN model for prediction of TMH that features by minimal parameter assumptions requirement and high computational efficiency. In the SOMPNN model, a self-organizing map (SOM) is used to adaptively learn the helices distribution knowledge hidden in the training data, and then a probabilistic neural network (PNN) is adopted to predict TMH segments based on the knowledge learned by SOM. Experimental results on two benchmark datasets show that the proposed SOMPNN outperforms most existing popular TMH predictors and is promising to be extended to deal with other complicated biological problems. The datasets and the source codes of SOMPNN are available at http://www.csbio.sjtu.edu.cn/bioinf/SOMPNN/.

Membrane protein integration into the endoplasmic reticulum

Most integral membrane proteins are targeted, inserted and assembled in the endoplasmic reticulum membrane. The sequential and potentially overlapping events necessary for membrane protein integration take place at sites termed translocons, which comprise a specific set of membrane proteins acting in concert with ribosomes and, probably, molecular chaperones to ensure the success of the whole process. In this minireview, we summarize our current understanding of helical membrane protein integration at the endoplasmic reticulum, and highlight specific characteristics that affect the biogenesis of multispanning membrane proteins.

Amino acid interaction preferences in helical membrane proteins

Membrane proteins are involved in a number of important biological functions. Yet, they are poorly understood from the structure and folding point of view. The external environment being drastically different from that of globular proteins, the intra-protein interactions in membrane proteins are also expected to be different. Hence, statistical potentials representing the features of inter-residue interactions based exclusively on the structures of membrane proteins are much needed. Currently, a reasonable number of structures are available, making it possible to undertake such an analysis on membrane proteins. In this study we have examined the inter-residue interaction propensities of amino acids in the membrane spanning regions of the alpha-helical membrane (HM) proteins. Recently we have shown that valuable information can be obtained on globular proteins by the evaluation of the pair-wise interactions of amino acids by classifying them into different structural environments, based on factors such as the secondary structure or the number of contacts that a residue can make. Here we have explored the possible ways of classifying the intra-protein environment of HM proteins and have developed scoring functions based on different classification schemes. On evaluation of different schemes, we find that the scheme which classifies amino acids to different intra-contact environment is the most promising one. Based on this classification scheme, we also redefine the hydrophobicity scale of amino acids in HM proteins.

Spatial structure and dimer–monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles

In present work the interaction of two TM α-helices of the ErbB3 receptor tyrosine kinase from the ErbB or HER family (residues 639–670) was studied by means of NMR spectroscopy in a membrane-mimicking environment provided by the DPC micelles. The ErbB3 TM segment appeared to form a parallel symmetric dimer in a left-handed orientation. The interaction between TM spans is accomplished via the non-standard motif and is supported by apolar contacts of bulky side chains and by stacking of aromatic rings together with π–cation interactions of Phe and Arg side chains. The investigation of the dimer–monomer equilibrium revealed thermodynamic properties of the assembly and the presence of two distinct regimes of the dimerization at low and at high peptide/detergent ratio. It was found that the detergent in case of ErbB3 behaves not as an ideal solvent, thus affecting the dimer–monomer equilibrium. Such behavior may account for the problems occurring with the refolding and stability of multispan helical membrane proteins in detergent solutions. The example of ErbB3 allows us to conclude that the thermodynamic parameters of dimerization, measured in micelles for two different helical pairs, cannot be compared without the investigation of their dependence on detergent concentration.

Membrane Protein Structure Determination using Paramagnetic Tags

The combination of paramagnetic tagging strategies with NMR or EPR spectroscopic techniques can revolutionize de novo structure determination of helical membrane proteins. Leveraging the full potential of this approach requires optimal labeling strategies and prediction of membrane protein topology from sparse and low-resolution distance restraints, as addressed by Chen etal. (2011).

Statistical analysis and exposure status classification of transmembrane beta barrel residues

Abstract Several computational methods exist for the identification of transmembrane beta barrel proteins (TMBs) from sequence. Some of these methods also provide the transmembrane (TM) boundaries of the putative TMBs. The aim of this study is to (1) derive the propensities of the TM residues to be exposed to the lipid bilayer and (2) to predict the exposure status (i.e. exposed to the bilayer or hidden in protein structure) of TMB residues. Three novel propensity scales namely, BTMC, BTMI and HTMI were derived for the TMB residues at the hydrophobic core region of the outer membrane (OM), the lipid–water interface regions of the OM, and for the helical membrane proteins (HMPs) residues at the lipid–water interface regions of the inner membrane (IM), respectively. Separate propensity scales were derived for monomeric and functionally oligomeric TMBs. The derived propensities reflect differing physico-chemical properties of the respective membrane bilayer regions and were employed in a computational method for the prediction of the exposure status of TMB residues. Based on the these propensities, the conservation indices and the frequency profile of the residues, the transmembrane residues were classified into buried/exposed with an accuracy of 77.91% and 80.42% for the residues at the membrane core and the interface regions, respectively. The correlation of the derived scales with different physico-chemical properties obtained from the AAIndex database are also discussed. Knowledge about the residue propensities and burial status will be useful in annotating putative TMBs with unknown structure.

Pre-Insertion Topology of Transmembrane Proteins is Highly Plastic and Can Be Controlled by a Single C-Terminal Residue

The mechanism by which helical membrane proteins are inserted into the cellular membrane remains unclear. It is known that membrane proteins are inserted co-translationally into the lipid bilayer and that positively charged residues in the loop regions of TM proteins are important topological determinants. However, it is unclear whether those charges act strictly locally-- affecting only the nearest transmembrane helices-- or act globally-- affecting the topology of the entire protein. We have found that the topology of an Escherichia coli inner membrane protein with four or five transmembrane helices can be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C-terminus. Addition of a charged, cleavable signal peptide to the N-terminus of the protein indicates that topology does not change after insertion into the membrane. These observation point to an unanticipated plasticity in pre-insertion membrane protein topology.

Transmembrane Helical Protein Folding: Lipid Modulation and Folding Transition States

General folding principles have emerged from studies on water-soluble proteins, but it is unclear how these ideas will translate to transmembrane proteins, which expose rather than hide their hydrophobic surfaces. We combine kinetic and thermodynamic studies of the reversible unfolding of helical membrane proteins to provide a definitive value for the reaction free energy and a means to probe the transition state. Efficient systems also need to be developed to stabilise, unfold and re-fold a wider range of alpha helical membrane proteins. We have been developing in vitro lipid bicelle and bilayer folding systems for membrane proteins. Bicelle properties, as well as the stored curvature elastic stress of model bilayers can be used to optimise the rate, yield and stability of folded protein. We have shown that events such as transmembrane helix insertion, as well as tertiary and quaternary structure formation are altered by the stored curvature stress of the bilayer. Either changing the lipid chains or introducing a different headgroup such as phosphoethanolamine alters the curvature stress of a phosphatoidylcholine lipid bilayer. Our results on a variety of monomeric, multidomain and multi-subunit proteins will be described.

Analyzing Inter-Residue Interactions for Structure Modeling of Helical Membrane Proteins

Transmembrane (TM) proteins are estimated to account for ∼20-30% of the human genome and serve as important drug targets. Despite the significant progress in experimental techniques, TM protein structure determination remains a challenge in general. Computational approaches, including de novo and comparative structure predictions, have played a significant role in structural and functional studies of membrane proteins, as well as in their structure-based drug design efforts. A major challenge of membrane protein structure prediction is to assemble individual helices into high-quality tertiary structures. Analyzing inter-residue interactions within membrane proteins is of significance to developing novel tools to tackle this challenge. In this work, we first identified four approaches for determining inter-residue interactions in protein structures and studied their correlation to individual quality measures. It was found that the best correlation was achieved by the approach focusing exclusively on favorable inter-residue interactions. Next, this best approach was applied to the analysis of multiple datasets of helical membrane protein structure models, including a dataset of homologous structure pairs with the sequence identity at the twilight zone. A simple measure was elucidated that directly correlates with the quality of these structure models. These results suggest a useful tool for computational modeling of membrane protein structures. This work was supported by the grant (1R15GM084404) from National Institute of Health.

Improved Helix and Kink Characterization in Membrane Proteins Allows Evaluation of Kink Sequence Predictors

Although the α-helical secondary structure of proteins is well-defined, the exact causes and structures of helical kinks are not. This is especially important for transmembrane (TM) helices of integral membrane proteins, many of which contain kinks providing functional diversity despite predominantly helical structure. We have developed a Monte Carlo method based algorithm, MC-HELAN, to determine helical axes alongside positions and angles of helical kinks. Analysis of all nonredundant high-resolution α-helical membrane protein structures (842 TM helices from 205 polypeptide chains) revealed kinks in 64% of TM helices, demonstrating that a significantly greater proportion of TM helices are kinked than those indicated by previous analyses. The residue proline is over-represented by a factor >5 if it is two or three residues C-terminal to a bend. Prolines also cause kinks with larger kink angles than other residues. However, only 33% of TM kinks are in proximity to a proline. Machine learning techniques were used to test for sequence-based predictors of kinks. Although kinks are somewhat predicted by sequence, kink formation appears to be driven predominantly by other factors. This study provides an improved view of the prevalence and architecture of kinks in helical membrane proteins and highlights the fundamental inaccuracy of the typical topological depiction of helical membrane proteins as series of ideal helices.

Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation

An understanding of the folding states of α-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in nuclear magnetic resonance spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation, we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.