Alpha-helical Membrane Proteins Research Articles

TmAlphaFold database: membrane localization and evaluation of AlphaFold2 predicted alpha-helical transmembrane protein structures.

AI-driven protein structure prediction, most notably AlphaFold2 (AF2) opens new frontiers for almost all fields of structural biology. As traditional structure prediction methods for transmembrane proteins were both complicated and error prone, AF2 is a great help to the community. Complementing the relatively meager number of experimental structures, AF2 provides 3Dpredictions for thousands of new alpha-helical membrane proteins. However, the lack of reliable structural templates and the fact that AF2 was not trained to handle phase boundaries also necessitates a delicate assessment of structural correctness. In our new database, Transmembrane AlphaFold database (TmAlphaFold database), we apply TMDET, a simple geometry-based method to visualize the likeliest position of the membrane plane. In addition, we calculate several parameters to evaluate the location of the protein into the membrane. This also allows TmAlphaFold database to show whether the predicted 3D structure is realistic or not. The TmAlphaFold database is available at https://tmalphafold.ttk.hu/.

Methods to study folding of alpha-helical membrane proteins in lipids.

How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.

How lipids affect the energetics of co-translational alpha helical membrane protein folding.

Membrane proteins need to fold with precision in order to function correctly, with misfolding potentially leading to disease. The proteins reside within a hydrophobic lipid membrane and must insert into the membrane and fold correctly, generally whilst they are being translated by the ribosome. Favourable and unfavourable free energy contributions are present throughout each stage of insertion and folding. The unfavourable energy cost of transferring peptide bonds into the hydrophobic membrane interior is compensated for by the favourable hydrophobic effect of partitioning a hydrophobic transmembrane alpha-helix into the membrane. Native membranes are composed of many different types of lipids, but how these different lipids influence folding and the associated free energies is not well understood. Altering the lipids in the bilayer is known to affect the probability of transmembrane helix insertion into the membrane, and lipids also affect protein stability and can promote successful folding. This review will summarise the free energy contributions associated with insertion and folding of alpha helical membrane proteins, as well as how lipids can make these processes more or less favourable. We will also discuss the implications of this work for the free energy landscape during the co-translational folding of alpha helical membrane proteins.

Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein.

The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome. We summarise the benefits of this approach and show how it can be successfully extended to a membrane protein with a complex topology. The bacterial leucine transporter, LeuT can be synthesised and inserted into lipid membranes using a variety of in vitro transcription translation systems. Unlike major facilitator superfamily transporters, where changes in lipids can optimise the amount of correctly inserted protein, LeuT insertion yields are much less dependent on the lipid composition. The presence of a bacterial translocon either in native membrane extracts or in reconstituted membranes also has little influence on the yield of LeuT incorporated into the lipid membrane, except at high reconstitution concentrations. LeuT is considered a paradigm for neurotransmitter transporters and possesses a knotted structure that is characteristic of this transporter family. This work provides a method in which to probe the formation of a protein as the polypeptide chain is being synthesised on a ribosome and inserting into lipids. We show that in comparison with the simpler major facilitator transporter structures, LeuT inserts less efficiently into membranes when synthesised cell-free, suggesting that more of the protein aggregates, likely as a result of the challenging formation of the knotted topology in the membrane.

Refining pairwise sequence alignments of membrane proteins by the incorporation of anchors.

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution, and in the generation of homology-based models. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly matched by a standard pairwise sequence alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the aligned regions either side of the newly matched positions. Previous studies have introduced restraints as a means to impose the matching of positions during sequence alignments, originally in the context of genome assembly. Here we introduce position restraints, or "anchors" as a feature in our alignment tool AlignMe, providing an aid to pairwise global sequence alignment of alpha-helical membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of anchors can be used to modify alignments, while still maintaining the reproducibility and rigor of the rest of the alignment. Anchored alignments can be generated using the online version of AlignMe available at www.bioinfo.mpg.de/AlignMe/.

Database Study on the Expression and Purification of Membrane Proteins.

Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio-function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta- barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

Virus-Host Protein Interactions in the Degradation Pathway of Human Serinc5 by NEF

Serine Incorporators (Serinc) are promising HIV therapeutic targets because of their ability to impair virus-target cell fusion. These all alpha-helical membrane proteins contain ten transmembrane segments, have limited extra-membranous domains, and are conserved in all eukaryotes. Human Serinc5 and Serinc3 inhibit HIV infectivity by impairing virus-target cell fusion through a proposed interaction with the HIV Env glycoprotein. However, Nef, an HIV auxiliary protein, counteracts Serinc's inhibition of membrane fusion by targeting it for degradation via clathrin-mediated endocytosis.

Investigating YidC-assisted Folding Pathways of Different Alpha-Helical Membrane Proteins

Insertion and folding of multi-spanning alpha-helical membrane proteins into the inner membrane of bacteria are crucial steps during protein biogenesis. These processes are assisted by a universally conserved machinery, the YidC insertase. It has been recently shown that YidC initiates folding of the lactose permease LacY by inserting a single structural segment into the membrane. Further folding of the LacY polypeptide occurs along variable trajectories until it reaches its native structure. However, to understand how YidC assists the insertion and folding of other membrane proteins, we first mechanically extract alpha-helical proteins, namely BetT3 and MelB, from a membrane using single-molecule force spectroscopy and record their characteristic unfolding spectra.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

Alpha-Helical Membrane Protein Folding in “Mixed” and “Ideal” Bicelles

Lipid-detergent bicelles are often used in membrane protein studies as an alternative to micelles. The ideal bicelle model contains a segregated lipid core surrounded by a detergent rim, and their morphologies depend on the ratio of lipid to detergent (q-ratio). There are several reports that the most widely used bicelle, containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, forms ideally mixed micelles at q-ratios below 0.5. To date, almost all membrane protein studies using isotropic bicelles, particularly solution NMR spectroscopy, have relied on q-ratios of ∼ 0.3 due to favorable tumbling properties. Designing bicelle mixtures with segregated lipid and detergent across a broad range of q-ratios is, therefore, of high interest for membrane protein applications. We investigated common bicelle mixtures with q-ratios of 0.0-1.0 and measured the short diameter, L, with SAXS. The diameter was previously shown to correspond with the mixing behavior reported by SANS and molecular dynamics simulations, with L equal to two lipid lengths in a lipid-segregated bicelle. All alkyl-chain lipid and detergent combinations with phosphocholine-like head groups form mixed micelles up to ∼ q-ratio = 0.5. Bicelles containing bulky moieties at detergent alkyl tails and head groups were additionally investigated, which we hypothesize will increase segregation between lipid and detergent components at q ratios less than 0.5. The mixtures most promising for ideal bicelle morphologies at low q-ratios were then used to investigate protein fold and backbone dynamics of a polytopic alpha-helical transmembrane protein using continuous-wave EPR and solution NMR spectroscopy. Our results suggest that lipid-protein interactions influence protein fold distinct from detergent-protein interactions.

Integrated Structural Biology for Alpha-Helical Membrane Protein Structure Determination

While great progress has been made, only 10% of the nearly 1,000 integral, a-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. We developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. We present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as lowas 1.2 A. We further illustrate application of these technologies for GPCR structure determination.

Impact of Lipid-Protein Interactions on Alpha-Helical Membrane Protein Fold

Impact of Lipid-Protein Interactions on Alpha-Helical Membrane Protein Fold

Lipids modulate the insertion and folding of the nascent chains of alpha helical membrane proteins.

Membrane proteins must be inserted into a membrane and folded into their correct structure to function correctly. This insertion occurs during translation and synthesis by the ribosome for most α-helical membrane proteins. Precisely how this co-translational insertion and folding occurs, and the role played by the surrounding lipids, is still not understood. Most of the work on the influence of the lipid environment on folding and insertion has focussed on denatured, fully translated proteins, and thus does not replicate folding during unidirectional elongation of nascent chains that occurs in the cell. This review aims to highlight recent advances in elucidating lipid composition and bilayer properties optimal for insertion and folding of nascent chains in the membrane and in the assembly of oligomeric proteins.

Investigating Cotranslational Folding in Membrane Proteins using Fragment-Based Structure Prediction

There is experimental evidence that both soluble and membrane proteins start to fold during the process of translation, while still attached to the ribosome. In the case of most alpha-helical membrane proteins, their insertion into the membrane is simultaneously carried out by the Sec complex. We have found evidence to suggest that membrane proteins are also forming tertiary interactions cotranslationally. We have tested two structure prediction protocols using our software SAINT2: one to imitate cotranslational folding which grows the nascent peptide from the N-terminus, and one to imitate in vitro folding which starts from an extended chain. The cotranslational method generates more accurate models, suggesting that it could be simulating the in vivo folding mechanism better. We are adapting the SAINT2 score to reflect the membrane environment in order to improve the accuracy of models produced and to more closely imitate in vivo folding. We are also exploring the effect of beginning from a segment of the correct native structure and using SAINT2 to complete the structure. This is to simulate a folding mechanism where the first part of the protein adopts part of its final fold even before the whole protein is synthesised.

Design Principles of Membrane Protein Structures

Membrane proteins play key role in cellular signaling and ion transport. Statistical analysis of expanding database of high-resolution membrane protein structures in Protein Data Bank (PDB) provides useful information about membrane protein structure and function. We used RosettaMembrane software (Yarov-Yarovoy V et al (2006) Proteins) to analyze ∼300 unique alpha helical membrane protein structures in PDB and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. The RosettaMembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state - from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. Residue buried state is determined from the number of residue neighbors within 6 and 10 A spheres. The RosettaMembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. Results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. We find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. Small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. Aromatic or positively charged amino acids and favorable facing the lipid head groups. Residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. These data will be useful for rational design of novel membrane protein structures and functions.

Development of a machine learning method to predict membrane protein-ligand binding residues using basic sequence information.

Locating ligand binding sites and finding the functionally important residues from protein sequences as well as structures became one of the challenges in understanding their function. Hence a Naïve Bayes classifier has been trained to predict whether a given amino acid residue in membrane protein sequence is a ligand binding residue or not using only sequence based information. The input to the classifier consists of the features of the target residue and two sequence neighbors on each side of the target residue. The classifier is trained and evaluated on a nonredundant set of 42 sequences (chains with at least one transmembrane domain) from 31 alpha-helical membrane proteins. The classifier achieves an overall accuracy of 70.7% with 72.5% specificity and 61.1% sensitivity in identifying ligand binding residues from sequence. The classifier performs better when the sequence is encoded by psi-blast generated PSSM profiles. Assessment of the predictions in the context of three-dimensional structures of proteins reveals the effectiveness of this method in identifying ligand binding sites from sequence information. In 83.3% (35 out of 42) of the proteins, the classifier identifies the ligand binding sites by correctly recognizing more than half of the binding residues. This will be useful to protein engineers in exploiting potential residues for functional assessment.

Computer Simulations for Predicting Membrane Protein Structures with the Replica-Exchange Methods and Implicit Membrane Model of a Restricted Configurational Space

The structures of membrane proteins are necessary to examine the functions and the mechanisms. The determination of membrane proteins takes a long time yet despite the development of experimental techniques. Thus, we have developed simulation methods for predicting alpha-helical membrane proteins. For the purpose, replica-exchange methods (REM) and a particular implicit membrane model are used. Distortions and kinked helix structures in transmembrane helices are frequently observed as a characteristic appearance of experimental membrane protein structures. Concerted rotation of torsion angles and dihedral angle of main chains in Monte Carlo move sets are implemented for including the distortions. Our implicit membrane model is to mimic the sampled configuration during native folding of membrane proteins after inserted membrane environment. We applied this method to bacteriorhodopsin, which has seven distorted transmembrane helices. From the random ideal helix configuration, we obtained local-minimum free energy states by REM simulations and principal component analysis. The RMSD value of whole backbone atoms from the PDB structure is 2.5 angstroms. The RMSD values in each helix structure about distortions are also less than about 1.5 angstroms.View Large Image | View Hi-Res Image | Download PowerPoint Slide

High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning

Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the study of membrane proteins in membrane mimetic and native membrane environments. These experiments often suffer from low sensitivity, due in part to the long recycle delays required for magnetization and probe recovery, as well as detection of low gamma nuclei. In ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work we investigate the sensitivity of (13)C and (1)H detected experiments applied to 27 kDa membrane proteins reconstituted in lipids and packed in small 1.3 mm MAS NMR rotors. We demonstrate that spin diffusion is sufficient to uniformly distribute paramagnetic relaxation enhancement provided by either covalently bound or dissolved CuEDTA over 7TM alpha helical membrane proteins. Using paramagnetic enhancement and low power decoupling in carbon detected experiments we can recycle experiments ~13 times faster than under traditional conditions. However, due to the small sample volume the overall sensitivity per unit time is still lower than that seen in the 3.2 mm probe. Proton detected experiments, however, showed increased efficiency and it was found that the 1.3 mm probe could achieve sensitivity comparable to that of the 3.2 mm in a given amount of time. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time.

Graph representation of high-dimensional alpha-helical membrane protein data

BackgroundIn genomics and proteomics, membrane protein analysis have shown that such analyses are very important to support the understanding of complex biological processes. In Genome-wide investigations of membrane proteins a large number of short, distinct sequence motifs has been revealed. Such motifs found so far support the understanding of the folded membrane protein in the membrane environment. They provide important information about functional or stabilizing properties. Recently several integrative approaches have been proposed to extract meaningful information out of the membrane environment. However, many information based approaches deliver results having deficits of visualisation outputs. Outgoing from high-throughput protein data analysis, these outputs play an important role in the evaluation of high-dimensional protein data, to establish a biological relationship and ultimately to provide useful information for research.ResultsWe have evaluated different resulting graphs generated from statistical analysis of consecutive motifs in helical structures of the membrane environment. Our results show that representative motifs with high occurrence in all investigated protein families are responsible for the general importance in alpha-helical membrane structure formation. Further, motifs which often occur with others in their function as so called “hubs” lead to the assumption, that these motifs constitute as important components in helical structures within the membrane. Otherwise, consecutive motifs and hubs which show a high occurrence in certain families only can be classified as important for family-specific functional characteristics. Summarized, we are able to bridge our graphical results from high-throughput analysis of membrane proteins over networking with databases to a biological context.ConclusionsOur results and the corresponding graphical visualisation support the understanding and interpretation of structure forming and functional motifs of membrane proteins. Our results are useful to interpret and refine results of common developed approaches. At last we show a simple way to visualise high-dimensional protein data in context to biological relevant information.

Phasing statistics for alpha helical membrane protein structures

In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno-L-methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data.