Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

A 10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are located in a stable helical region of a transmembrane peptide. The 25-residue peptide (sMTM7) used mimics the cytoplasmic proton hemichannel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The three-dimensional structure of peptide sMTM7 in DMSO has been previously solved by NMR spectroscopy. The radial and spatial distributions of the DMSO molecules surrounding the peptide as well as the number of hydrogen bonds between DMSO and the side chains of the amino acid residues involved are extracted from the molecular dynamics simulations. Analysis of the molecular dynamics trajectories shows that the amino acid side chains are fully embedded in DMSO. Polar and positively charged amino acid side chains have dipole-dipole interactions with the oxygen atom of DMSO and form hydrogen bonds. Apolar residues become solvated by DMSO through the formation of a hydrophobic pocket in which the methyl groups of DMSO are pointing toward the hydrophobic side chains of the residues involved. The dual solvation properties of DMSO cause it to be a good membrane-mimicking solvent for transmembrane peptides that do not unfold due to the presence of DMSO.     

Identification of photolabeled peptides for the acceptor substrate binding domain of beta 1,4-galactosyltransferase.

We successfully applied a carbene-generating N-acetylglucosamine derivative carrying a biotinyl group to the radioisotope-free identification of peptides within bovine UDP-galactose: N-acetylglucosamine beta 1,4-galactosyltransferase (GalT, EC 2.4.1.38) catalytic domain. Owing to the low yield of cross-linking, conventional photoaffinity labeling experiments usually encounter a thorny problem in attempting to isolate labeled components from very complex mixtures. A biotin tag introduced with our photoaffinity probe enabled us to separate the photolabeled protein from a large amount of coexisting unlabeled GalT. The introduction of biotin was also useful for the radioisotope-free detection of a labeled protein based on a highly sensitive chemiluminescent technique. We developed a novel poly(vinylidene difluoride) membrane for the identification of labeled peptides in a simple dot blot assay. Using this membrane, we successfully identified biotinyl peptides among a number of HPLC separated fragments derived from the protease digestion of photolabeled GalT proteins. The sequence analysis revealed that the biotin tag was incorporated within a tryptic GalT fragment of Y197-R208. Our approach yields, for the first time, information on the acceptor substrate binding-site fragment in this enzyme, that has been difficult to obtain using other approaches. These data are consistent with previous suggestions concerning the GalT acceptor site and clearly demonstrate the effectiveness of our approach for rapid identification of photolabeled peptides.     

A ruler for determining the position of proteins in membranes

Both the oxygen diffusion rate and the oxygen solubility vary with depth into the interior of biological membranes. The product of these two gradients generates a single gradient, a permeability gradient, which is a smooth continuous function of the distance from the center of the membrane. Using electron paramagnetic resonance and the spin-probe method, the relaxation gradient of oxygen, which is directly proportional to the permeability gradient, is the quantity that can be directly measured in membranes under physiological conditions. The gradient obtained provides a calibrated ruler for determining the membrane depth of residues either from loop regions of membrane-binding proteins or from the membrane-exposed residues of transmembrane proteins. We have determined the relaxation gradient of oxygen in zwitterionic and anionic phospholipid membranes by attaching a single nitroxide probe to a transmembrane alpha-helical polypeptide at specific residues. The peptide ruler was used to determine the depth of penetration of the calcium-binding loops of the C2 domain of cytosolic phospholipase A(2). The positions of selected residues of this membrane-binding protein that penetrate into the membrane, determined using this ruler, compared favorably with previous determinations using more complex methods. The relaxation gradient constrains the possible values of the membrane-dependent oxygen concentration and the oxygen diffusion gradients. The average oxygen diffusion coefficient is estimated to be at least 2-fold smaller in the membrane than that in water.     

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.     

Effect of transmembrane Ca2+ gradient on conformation and enzyme activity of reconstituted adenylate cyclase.

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca2+ gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca2+ concentration (approximately 10(-6) mol/L, similar to the physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. outside higher Ca2+ concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca2+ ionophore A23187. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. The conformation difference of adenylate cyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

Neural network-based prediction of transmembrane beta-strand segments in outer membrane proteins

Prediction of transmembrane beta-strands in outer membrane proteins (OMP) is one of the important problems in computational chemistry and biology. In this work, we propose a method based on neural networks for identifying the membrane-spanning beta-strands. We introduce the concept of "residue probability" for assigning residues in transmembrane beta-strand segments. The performance of our method is evaluated with single-residue accuracy, correlation, specificity, and sensitivity. Our predicted segments show a good agreement with experimental observations with an accuracy level of 73% solely from amino acid sequence information. Further, the predictive power of N- and C-terminal residues in each segments, number of segments in each protein, and the influence of cutoff probability for identifying membrane-spanning beta-strands will be discussed. We have developed a Web server for predicting the transmembrane beta-strands from the amino acid sequence, and the prediction results are available at http://psfs.cbrc.jp/tmbeta-net/.     

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.     

Prediction of number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone

Prediction of protein domain boundaries is an important step for the prediction of three-dimensional structure. The simple method PDP has been elaborated for prediction of the number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone. The method uses an optimized scale based on the statistics of appearance of amino acid residues at domain boundaries. Our method demonstrates promising results in comparison to other methods that do not use homologous sequences. From the database of proteins that are targets from CASP6 (Critical Assessment of Techniques for Protein Structure Prediction) our program correctly assigned the number of domains for approximately 80% of one domain proteins and approximately 50% for two-domain proteins. Our method offers three main advantages: it is very simple, it is fast, and it uses a minimal number of parameters in comparison with other methods.     

Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04+/-0.02 A(-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15+/-0.05 A(-1)) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.     

酶切过程中肽段过烷基化对蛋白质定性和定量分析的影响

蛋白质的还原-烷基化是蛋白质酶切中的重要步骤,常用的烷基化试剂是碘乙酰胺(IAA),但是IAA除了和半胱氨酸发生反应,也可能和其他多种氨基酸发生副反应。我们模拟常规的酶切条件,系统地研究了蛋白质真实酶切时所有酶切肽段发生烷基化的情况。结果表明,多种氨基酸可以发生烷基化,其趋势为:半胱氨酸>肽段N端氨基酸>天冬氨酸>谷氨酸>组氨酸>天冬酰胺>赖氨酸>酪氨酸,同时也发现同一肽段上的氨基酸烷基化具有排他性和聚集性。根据定性结果,采用质谱多反应监测(MRM)技术对多个肽段进行了定量分析,评估了过烷基化对蛋白质定量分析的影响。该研究结果表明,过量的烷基化修饰对蛋白质的定性与定量分析都可能产生较大影响。在蛋白质组学研究的样本处理流程中,应避免样本的过烷基化。     

Modeling anesthetic binding sites within the glycine alpha one receptor based on prokaryotic ion channel templates: the problem with TM4

Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.     

Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.     

Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein.     

beta-Barrel transmembrane proteins: Geometric modelling, detection of transmembrane region, and structural properties

The location of the membrane lipid bilayer relative to a transmembrane protein structure is important in protein engineering. Since it is not present on the determined structures, it is essential to automatically define the membrane embedded protein region in order to test mutation effects or to design potential drugs. beta-Barrel transmembrane proteins, present in nature as outer membrane proteins (OMPs), comprise one of the two transmembrane protein fold classes. Lately, the number of their determined structures has increased and this enables the implementation and evaluation of structure-based annotation methods and their more comprehensive study. In this paper, we propose two new algorithms for (i) the geometric modelling of beta-barrels and (ii) the detection of the transmembrane region of a beta-barrel transmembrane protein. The geometric modelling algorithm combines a non-linear least square minimization method and a genetic algorithm in order to find the characteristics (axis, radius) of a shape with axial symmetry which best models a beta-barrel. The transmembrane region is detected by profiling the external residues of the beta-barrel along its axis in terms of hydrophobicity and existence of aromatic and charged residues. TbB-Tool implements these algorithms and is available in . A non-redundant set of 22 OMPs is used in order to evaluate the algorithms implemented and the results are very satisfying. In addition, we quantify the abundance of all amino acids and the average hydrophobicity for external and internal beta-stranded residues along the axis of beta-barrel, thus confirming and extending other researchers' results.     

The prediction of amphiphilic alpha-helices

A number of sequence-based analyses have been developed to identify protein segments, which are able to form membrane interactive amphiphilic alpha-helices. Earlier techniques attempted to detect the characteristic periodicity in hydrophobic amino acid residues shown by these structure and included the Molecular Hydrophobic Potential (MHP), which represents the hydrophobicity of amino acid residues as lines of isopotential around the alpha-helix and analyses based on Fourier transforms. These latter analyses compare the periodicity of hydrophobic residues in a putative alpha-helical sequence with that of a test mathematical function to provide a measure of amphiphilicity using either the Amphipathic Index or the Hydrophobic Moment. More recently, the introduction of computational procedures based on techniques such as hydropathy analysis, homology modelling, multiple sequence alignments and neural networks has led to the prediction of transmembrane alpha-helices with accuracies of the order of 95% and transmembrane protein topology with accuracies greater than 75%. Statistical approaches to transmembrane protein modeling such as hidden Markov models have increased these prediction levels to an even higher level. Here, we review a number of these predictive techniques and consider problems associated with their use in the prediction of structure / function relationships, using alpha-helices from G-coupled protein receptors, penicillin binding proteins, apolipoproteins, peptide hormones, lytic peptides and tilted peptides as examples.     

白喉毒素活性中心的量子化学计算与149位突变体的酶学动力学

通过量子化学计算确定白喉毒素分子催化区活性中心的关键氨基酸残基, 评价其取代后的酶活性的改变, 为导向性抗癌药物研究提供高效杀伤细胞工具. 结合目前关于白喉毒素结构与功能的研究状况和量子化学计算结果, 将白喉毒素催化区的第149位酪氨酸突变为苯丙氨酸, 对其酶活性和与底物的结合能力进行评价. Y149位酪氨酸位于正电中心, 起受电子作用, 与野生白喉毒素相比, 苯丙氨酸突变体的酶催化活性增加约一倍, 而与底物结合能力没有变化. Y149是酶活性中心的关键氨基酸残基, 对其取代能够影响蛋白质的生物活性.     

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for cytochrome c, and 9 to 8 Å for β-2-microglobulin.     

Ion‐Channel Gating in Transmembrane Receptor Proteins: Functional Activity in Tethered Lipid Membranes

Through thiolipids a planar lipid bilayer (1) was immobilized on a gold support (2) for use as an electrode. This allows the detection of the ligand‐gating function of the natural transmembrane channel protein OmpF (3) reconstituted in the artificial membrane: the binding of a domain (4) of the toxin colicin N, observed by surface plasmon resonance, induces the blocking of the OmpF channel protein, as shown by impedance spectroscopy.     

Structure determination of a membrane protein in proteoliposomes

An NMR method for determining the three-dimensional structures of membrane proteins in proteoliposomes is demonstrated by determining the structure of MerFt, the 60-residue helix-loop-helix integral membrane core of the 81-residue mercury transporter MerF. The method merges elements of oriented sample (OS) solid-state NMR and magic angle spinning (MAS) solid-state NMR techniques to measure orientation restraints relative to a single external axis (the bilayer normal) from individual residues in a uniformly (13)C/(15)N labeled protein in unoriented liquid crystalline phospholipid bilayers. The method relies on the fast (>10(5) Hz) rotational diffusion of membrane proteins in bilayers to average the static chemical shift anisotropy and heteronuclear dipole-dipole coupling powder patterns to axially symmetric powder patterns with reduced frequency spans. The frequency associated with the parallel edge of such motionally averaged powder patterns is exactly the same as that measured from the single line resonance in the spectrum of a stationary sample that is macroscopically aligned parallel to the direction of the applied magnetic field. All data are collected on unoriented samples undergoing MAS. Averaging of the homonuclear (13)C/(13)C dipolar couplings, by MAS of the sample, enables the use of uniformly (13)C/(15)N labeled proteins, which provides enhanced sensitivity through direct (13)C detection as well as the use of multidimensional MAS solid-state NMR methods for resolving and assigning resonances. The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes.