Thoeretical study of the packing of α-helices of poly(l-alanine) into transmembrane bundles. Possible significance for ion-transfer

Energy optimizations are carried out on packages of Nα-helices of poly(l-alanine) from N = 3−7, starting from an initial arrangement of the helices at the vertices of various polygonal prisms, in view of the possible formation of channel-making bundles in membranes. The results show: that, for each N, a number of stable packages exist; that the presence of one pair (and even two) of adjacent parallel helices in a package is not incompatible with its stability, due to the overcompensation of its unfavorable electrostatic energy by the sum of the corresponding favorable terms for the antiparallel pairs; and that some packages provide ready-made pores in their interior. The energy profile computed for Na⁺ inside one of the pores (resulting from five helices) shows a favorable energy all the way through, in spite of the methyl groups protruding into the channel. Similarly one water molecule interacts favorably with this pore throughout.     

Simulation of the packing of idealized transmembrane α-helix bundles

The aim of this study is to investigate if the packing motifs of native transmembrane helices can be produced by simulations with simple potentials and to develop a method for the rapid generation of initial candidate models for integral membrane proteins composed of bundles of transmembrane helices. Constituent residues are mapped along the helix axis in order to maintain the amino acid sequence-dependent properties of the helix. Helix packing is optimized according to a semi-empirical potential mainly composed of four components: a bilayer potential, a crossing angle potential, a helix dipole potential and a helix-helix distance potential. A Monte Carlo simulated annealing protocol is employed to optimize the helix bundle system. Necessary parameters are derived from theoretical studies and statistical analysis of experimentally determined protein structures. Preliminary testing of the method has been conducted with idealized seven Ala₂₀ helix bundles. The structures generated show a high degree of compactness. It was observed that both bacteriorhodopsin-like and δ-endotoxin-like structures are generated in seven-helix bundle simulations, within which the composition varies dependent upon the cooling rate. The simulation method has also been employed to explore the packing of N = 4 and N = 12 transmembrane helix bundles. The results suggest that seven and 12 transmembrane helix bundles resembling those observed experimentally (e.g., bacteriorhodopsin, rhodopsin and cytochrome c oxidase subunit I) may be generated by simulations using simple potentials. Received: 16 November 1998 / Revised version: 26 March 1999 / Accepted: 8 April 1999     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Transmembrane orientation of α-helices in the thylakoid membrane and in the light-harvesting complex. A polarized infrared spectroscopy study

The structure and orientation of the major protein constituent of photosynthetic membranes in green plants, the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) have been investigated by ultraviolet circular dichroism (CD) and polarized infrared spectroscopies. The isolated purified LHC has been reconstituted into phosphatidylcholine vesicles and has been compared to the pea thylakoid membrane. The native orientation of the pigments in the LHC reconstituted in vesicles was characterized by monitoring the low-temperature polarized absorption and fluorescence spectra of reconstituted membranes. Conformational analysis of thylakoid and LHC indicate that a large proportion of the thylakoid protein is in the α-helical structure (56 ± 4%), while the LHC is for 44 ± 7% α-helical. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented multilayers of thylakoids and LHC reconstituted in vesicles, we have estimated the degree of orientation of the α-helical chains with respect to the membrane normal. Infrared dichroism data demonstrate that transmembrane α-helices are present in both thylakoid and LHC with the α-helix axes tilted at less than 30° in LHC and 40° in thylakoid with respect to the membrane normal. In thylakoids, an orientation of the polar C=O ester groups of the lipids parallel to the membrane plane is detected. Our results are consistent with the existence of 3–5 transmembrane α-helical segments in the LHC molecules.     

Role of the structural context in selection of hydrophobic side-chain rotamers in a- and d-positions of α-helices

It was demonstrated for the first time that the distribution of side-chain rotamers in the a-and d-positions of α-helices of coiled-coil (cc) proteins follows a certain trend, rather then being random. For instance, most side chains adopt t rotamers in the a-positions and g^? rotamers in the d-positions of helical dimers. Vice versa, most side chains adopt g^? rotamers in the a-positions and t rotamers in the d-positions of tetramers. It was concluded that selection of the side-chain rotamers depends on the packing of α-helices and, consequently, depends on the structural context.     

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. ²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively ¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10^- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.     

The complete rate equation,including the explicit dependence on Na+ ions,for the influx of α-aminoisobutyric acid into mouse brain slices

Summary The rate equation, including dependence on Na⁺-ion concentration for the influx of -aminoisobutyric acid into mouse brain slices incubated in isotonic glucose medium at 37°C, isv=0.402S/{1.02(1+788/[Na⁺]²)+S}+0.0477S, wherev=influx in mol/min, g final wet wt of slices; [Na⁺]=concentration of Na⁺ ions in medium, inmm; andS=concentration of -aminoisobutyric acid in medium, inmm. This equation shows two kinetically independent, parallel pathways of concentrative uptake: one, saturable and dependent on Na⁺; the other, unsaturable and independent of Na⁺. Influx is independent of ionic strength, Cl^– ionper se, and a moderate increase in tonicity. The binding of substrate to the saturable carrier depends on the Na⁺ concentration; the maximum capacity of this carrier does not. For transport, 2 Na⁺ ions must interact with each saturable transport site. This does not imply coupling between the flux of Na⁺ and the flux of -aminoisobutyric acid.     

NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites

The α7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the α7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human α7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the α7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit α7 function, but the latter can. The α7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the α7 TM domain is smaller than the equivalent cavity in the α4β2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the α7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the α7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the α7 channel-gate residue L9′. Furthermore, halothane did not induce profound dynamics changes in the α7 channel as observed in α4β2. The study offers a novel high-resolution structure for the human α7 nAChR TM domain that is invaluable for developing α7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences.     

Studies on carbohydrate-metabolizing enzymes. The hydrolysis of α-glucosides, including nigerose, by extracts of alfalfa and other higher plants

1. Enzyme preparations from 11 plant sources, from yeast and from the protozoan Tetrahymena pyriformis show nigerase activity, which, in most preparations, was 70–90% of that towards maltose. 2. These enzyme preparations also hydrolysed isomaltose, but there was a wide variation in relative maltase to isomaltase activity. 3. The maltase and nigerase activities of alfalfa and tomato preparations could not be differentiated by heat inactivation or inhibitor methods. However, with turanose used as a competitive inhibitor, evidence suggesting that maltose and nigerose are hydrolysed at different catalytically active sites in the alfalfa preparation was obtained. 4. It is probable that the alfalfa α-glucosidase exists as a mixture of isoenzymes.     

Theoretical studies on the interaction of proteins and nucleic acid: II. The binding of α-helix to B-DNA

Interactions between B-DNA and homopolymeric α-helices of glycine, alanine, serine, asparagine and aspartic acid have been studied theoretically. The complexation energy has been minimised taking into account the interactions between DNA and the polypeptides as well as the internal energy of the α-helix and the interaction energy of counterions with the complex. The results obtained indicate the important role of strong hydrogen bonds between the peptide side chains and nucleic acid phosphate groups, these bonds being much stronger than specific interactions with the base-pairs. The formation of these structural bonds depends on the size of the α-helix, which in turn determines whether bridging across the major groove is possible. The steric role of the methyl group of thymine in orienting the peptide helix and the role of DNA screening cations in complex stabilization are also significant.     

Accurate prediction of the burial status of transmembrane residues of α-helix membrane protein by incorporating the structural and physicochemical features

Predicting the burial status (the residue exposure to the lipid bilayer or buried within the protein core) of transmembrane (TM) residues of α-helix membrane protein (αHMP) is of great importance for genome-wide annotation and for experimental researchers to elucidate diverse physiological processes. In this work, we developed a new computational model that can be used for predicting the burial status of TM residues of αHMP. By incorporating physicochemical scales and conservation index, an efficient prediction model using least squares support vector machine (LS-SVM) was developed. The model was developed from 43 protein chains and its prediction ability was evaluated by an independent test set of other non-redundant ten protein chains. The prediction accuracy of our method was much better than the results of the reported works. Our results demonstrate that the LS-SVM prediction model incorporating structural and physicochemical features derived from sequence information could greatly improve the prediction accuracy.     

Insights into the thermodynamics of copper association with amyloid-β, α-synuclein and prion proteins

This review examines recent studies on the thermodynamics of copper association with amyloid-β, α-synuclein and prion protein, with an eye towards using this information to understand the etiology of associated neurodegenerative diseases. A variety of binding affinities and binding sites, which are essential to understand the function and consequence of copper-protein interaction, have been reported for copper to these three neurobiologic systems. This current review reconciles the disparate models presented in the literature.     

Structural and thermodynamic insight into the process of “weak” dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix–helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure–function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651–678)₂ form a right-handed parallel dimer through the N-terminal double GG4-like motif A⁶⁵⁵GxxGG⁶⁶⁰ in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer–dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true (“ideal”) solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix–helix interactions, which may be critical for membrane protein activity.     

Synthesis and study of the α-amylase inhibitory potential of thiadiazole quinoline derivatives

α-Amylase is a target for type-2 diabetes mellitus treatment. However, small molecule inhibitors of α-amylase are currently scarce. In the course of developing small molecule α-amylase inhibitors, we designed and synthesized thiadiazole quinoline analogs (1−30), characterized by different spectroscopic techniques such as ¹HNMR and EI-MS and screened for α-amylase inhibitory potential. Thirteen analogs 1, 2, 3, 4, 5, 6, 22, 23, 25, 26, 27, 28 and 30 showed outstanding α-amylase inhibitory potential with IC₅₀ values ranges between 0.002 ± 0.60 and 42.31 ± 0.17 μM which is many folds better than standard acarbose having IC₅₀ value 53.02 ± 0.12 μM. Eleven analogs 7, 9, 10, 11, 12, 14, 15, 17, 18, 19 and 24 showed good to moderate inhibitory potential while seven analogs 8, 13, 16, 20, 21 and 29 were found inactive. Our study identifies novel series of potent α-amylase inhibitors for further investigation. Structure activity relationship has been established.     

Theoretical study of electronic absorptions in aminopyridines – TCNE CT complexes by quantum chemical methods, including solvent

The geometric and electronic structure of donor-acceptor complexes of TCNE with aniline, o-, m- and p- aminopyridines and pyridine has been studied in gas phase and in solution using CC2, TDDFT and CIS methods. Concerning interaction energy between particular donor and TCNE acceptor it is fairly described by both CC2 (MP2) and DFT-D approaches. Transition energies in gas phase calculated by CC2 approach are in good agreement with available experimental data for aniline. TDDFT calculations with LC-BLYP functional (with standard value of range separation factor μ?=?0.47) gives transition energies too high although not as high as CIS. The red solvent shifts, calculated by PCM model with CIS method are qualitative correct, but error in the range of 0.1-0.2 eV should be expected.     

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. This protocol describes an optimized synthetic strategy for the preparation of stabilized α-helices that feature a carbon-carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of canonical helices. Formation of the carbon-carbon bond is enabled by a microwave-assisted ring-closing metathesis reaction between two terminal olefins on the peptide chain. The outlined strategy allows the synthesis and purification of a hydrogen bond surrogate (HBS) α-helix in ～ 1 week.