Seven-helix Transmembrane Protein Research Articles

Molecular Biology of Microbial Rhodopsins.

Research on type 1 rhodopsins spans now a history of 50years. Originally, just archaeal ion pumps and sensors have been discovered. However, with modern genetic techniques and gene sequencing tools, more and more proteins were identified in all kingdoms of life. Spectroscopic and other biophysical studies revealed quite diverse functions. Ion pumps, sensors, and channels are imprinted in the same seven-helix transmembrane protein scaffold carrying a retinal prosthetic group. In this review, molecular biology methods are described, which enabled the elucidation of their function and structure leading to optogenetic applications.

Current-voltage characteristics of seven-helix proteins from a cubic array of amino acids.

The electrical properties of a set of seven-helix transmembrane proteins, whose space arrangement [three-dimensional (3D) structure] is known, are investigated by using regular arrays of the amino acids. These structures, specifically cubes, have topological features similar to those shown by the chosen proteins. The theoretical results show a good agreement between the predicted current-voltage characteristics obtained from a cubic array and those obtained from a detailed 3D structure. The agreement is confirmed by available experiments on bacteriorhodopsin. Furthermore, all the analyzed proteins are found to share the same critical behavior of the voltage-dependent conductance and of its variance. In particular, the cubic arrangement evidences a short plateau of the excess conductance and its variance at high voltages. The results of the present investigation show the possibility to predict the I-V characteristics of a multiple-protein sample even in the absence of detailed knowledge of the proteins' 3D structure.

Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins

Three dimers of the amphipathic α-helical peptide 18A have been synthesized with different interhelical linkers inserted between the two copies of 18A. The dimeric peptides were denoted 'beltides' where Beltide-1 refers to the 18A-dimer without a linker, Beltide-2 is the 18A-dimer with proline (Pro) as a linker and Beltide-3 is the 18A-dimer linked by two glycines (Gly-Gly). The self-assembly of the beltides with the phospholipid DMPC was studied with and without the incorporated membrane protein bacteriorhodopsin (bR) through a combination of coarse-grained MD simulations, size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, small-angle scattering (SAS), static light scattering (SLS) and UV-Vis spectroscopy. For all three beltides, MD and combined small-angle X-ray and -neutron scattering were consistent with a disc structure composed by a phospholipid bilayer surrounded by a belt of peptides and with a total disc diameter of approximately 10 nm. CD confirmed that all three beltides were α-helical in the free form and with DMPC. However, as shown by SEC the different interhelical linkers clearly led to different properties of the beltides. Beltide-3, with the Gly-Gly linker, was very adaptable such that peptide nanodiscs could be formed for a broad range of different peptide to lipid stoichiometries and therefore also possible disc-sizes. On the other hand, both Beltide-2 with the Pro linker and Beltide-1 without a linker were less adaptable and would only form discs of certain peptide to lipid stoichiometries. SLS revealed that the structural stability of the formed peptide nanodiscs was also highly affected by the linkers and it was found that Beltide-1 gave more stable discs than the other two beltides. With respect to membrane protein stabilization, each of the three beltides in combination with DMPC stabilizes the seven-helix transmembrane protein bacteriorhodopsin significantly better than the detergent octyl glucoside, but no significant difference was observed between the three beltides. We conclude that adaptability, size, and structural stability can be tuned by changing the interhelical linker while maintaining the properties of the discs with respect to membrane protein stabilization.

Crystal Structures of Activated G-Protein Coupled Receptors

G protein coupled receptors (GPCRs) are seven-helix transmembrane proteins that constitute the largest family of membrane proteins in the human genome. GPCRs are responsible for the majority of signal transduction events involving hormones and neurotransmitters across the cell membrane. Activating ligands (agonists) promote binding to and nucleotide exchange by a heterotrimeric G-protein, which dissociates and interacts with downstream effectors. GPCRs are highly dynamic structures, and for many years they resisted crystallization efforts. A number of methods, including protein engineering, lipid-based crystallization, and microfocus x-ray beamlines have enabled the recent explosion of GPCR structures. The majority of these structures are in the inactive state, and efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. I will discuss the strategies that have enabled crystal structure determination of the β2-adrenergic and M2 muscarinic receptors in their activated states, and discuss key features of the coupling between agonist binding and conformational changes that enable interaction with G proteins.

RpkA, a Highly Conserved GPCR with a Lipid Kinase Domain, Has a Role in Phagocytosis and Anti-Bacterial Defense

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA-. The difference between wild type and rpkA- was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA- we could not detect a difference in endosomal pH but rpkA- showed depletion of phosphoinositides (PIP and PIP2) when we compared metabolically labeled phosphoinositides from wild type and rpkA-. Furthermore rpkA- exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.

Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.

Comparison Analysis of Primary Ligand Binding Sites in Seven-Helical Membrane Proteins

Seven-helix transmembrane proteins, including the G-protein coupled receptors, mediate a broad range of fundamental cellular activities through binding to a wide range of ligands. Understanding the structural basis for the ligand-binding selectivity of these proteins is of significance to their structure-based drug design. Comparison analysis of proteins' ligand binding sites provides a useful way to study their structure-activity relationships. Various computational methods have been developed for the binding site comparison of soluble proteins. In this work, we applied this approach to the analysis of the primary ligand-binding sites of 92 seven-helix transmembrane proteins. Results of the studies confirmed that the binding site of bacterial rhodopsins is indeed different from all G-protein coupled receptors. In the latter group, further comparison of the binding sites indicated a group of residues that could be responsible for ligand-binding selectivity and important for structure-based drug design. Further, unexpected binding site dissimilarities were observed among adrenergic and adenosine receptors, suggesting that the percentage of the overall sequence identity between a target protein and a template protein alone is not sufficient for selecting the best template for homology modeling of seven-helix membrane proteins. These results provided novel insight into the structural basis of ligand-binding selectivity of seven-helix membrane proteins and are of practical use to the computational modeling of these proteins.

Comparison analysis of primary ligand‐binding sites in seven‐helix membrane proteins

Seven-helix transmembrane proteins, including the G-protein-coupled receptors (GPCRs), mediate a broad range of fundamental cellular activities through binding to a wide range of ligands. Understanding the structural basis for the ligand-binding selectivity of these proteins is of significance to their structure-based drug design. Comparison analysis of proteins' ligand-binding sites provides a useful way to study their structure-activity relationships. Various computational methods have been developed for the binding-site comparison of soluble proteins. In this work, we applied this approach to the analysis of the primary ligand-binding sites of 92 seven-helix transmembrane proteins. Results of the studies confirmed that the binding site of bacterial rhodopsins is indeed different from all GPCRs. In the latter group, further comparison of the binding sites indicated a group of residues that could be responsible for ligand-binding selectivity and important for structure-based drug design. Furthermore, unexpected binding-site dissimilarities were observed among adrenergic and adenosine receptors, suggesting that the percentage of the overall sequence identity between a target protein and a template protein alone is not sufficient for selecting the best template for homology modeling of seven-helix membrane proteins. These results provided novel insight into the structural basis of ligand-binding selectivity of seven-helix membrane proteins and are of practical use to the computational modeling of these proteins.

Identifying G-protein Coupled Receptors Using Weighted Levenshtein Distance and Nearest Neighbor Method

G-protein coupled receptors (GPCRs) are a class of seven-helix transmembrane proteins that have been used in bioinformatics as the targets to facilitate drug discovery for human diseases. Although thousands of GPCR sequences have been collected, the ligand specificity of many GPCRs is still unknown and only one crystal structure of the rhodopsin-like family has been solved. Therefore, identifying GPCR types only from sequence data has become an important research issue. In this study, a novel technique for identifying GPCR types based on the weighted Levenshtein distance between two receptor sequences and the nearest neighbor method (NNM) is introduced, which can deal with receptor sequences with different lengths directly. In our experiments for classifying four classes (acetylcholine, adrenoceptor, dopamine, and serotonin) of the rhodopsin-like family of GPCRs, the error rates from the leave-one-out procedure and the leave-half-out procedure were 0.62% and 1.24%, respectively. These results are prior to those of the covariant discriminant algorithm, the support vector machine method, and the NNM with Euclidean distance.

Selective proteome-wide detection of hydrophobic integral membrane proteins using a novel fluorescence-based staining technology.

Integral proteins containing two or more alpha-helical membrane-spanning domains are underrepresented in two-dimensional gels. While sodium dodecyl sulfate (SDS)-polyacrylamide gels separate these proteins, staining profiles are usually dominated by high-abundance hydrophilic proteins in the specimen. A fluorescence-based stain is presented that selectively highlights integral proteins containing two or more alpha-helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5-10 ng of bacteriorhodopsin, a seven-helix transmembrane protein. Stained proteins are detected using a laser scanner or charge-coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.

Is the olfactory receptor a metalloprotein?

The sense of smell is arguably our most primal faculty and also the least understood. Even our own olfactorily impaired species is capable of detecting approximately 10,000 distinct scents [Buck, L. & Axel, R. (1991) Cell 65, 175-187]. To achieve that amazing diversity, mammals have approximately 1,000 olfactory genes, which accounts for approximately 3% of their entire genome [Mombaerts, P. (1999) Science 286, 707-711]. The olfactory receptors (ORs) are believed to be seven-helix transmembrane proteins, with an odorant-binding site on the periplasmic domain and a G protein-binding site on the cytoplasmic domain. Odorants first bind to an OR, which then undergoes some structural change that triggers the G protein activation and the following cascade of events leading to nerve cell activity. The structural details of ORs, however, remain to be determined. In this paper, we will describe a hypothesis in which metal ions play an important role for odorant recognition. We analyze the predicted structure and consensus sequence of the ORs and propose a metal-binding site in the loop between fourth and fifth helix (4-5 loop). We have prepared synthetically a pentapeptide that contains this putative binding site and find that it not only has high affinity for binding Cu(II) and Zn(II) ions, but that it also undergoes a dramatic transition to an alpha-helical structure upon metal ion binding. Based on these observations, we propose a "shuttlecock" mechanism for the possible structural change in ORs upon odorant binding. This mechanism involves membrane penetration of the 4-5 loop after residue charge neutralization by metal ion binding.