Computational Insights into the Charge Relaying Properties of β‐Turn Peptides in Protein Charge Transfers

Density functional theory calculations suggest that β‐turn peptide segments can act as a novel dual‐relay elements to facilitate long‐range charge hopping transport in proteins, with the N terminus relaying electron hopping transfer and the C terminus relaying hole hopping migration. The electron‐ or hole‐binding ability of such a β‐turn is subject to the conformations of oligopeptides and lengths of its linking strands. On the one hand, strand extension at the C‐terminal end of a β‐turn considerably enhances the electron‐binding of the β‐turn N terminus, due to its unique electropositivity in the macro‐dipole, but does not enhance hole‐forming of the β‐turn C terminus because of competition from other sites within the β‐strand. On the other hand, strand extension at the N terminal end of the β‐turn greatly enhances hole‐binding of the β‐turn C terminus, due to its distinct electronegativity in the macro‐dipole, but does not considerably enhance electron‐binding ability of the N terminus because of the shared responsibility of other sites in the β‐strand. Thus, in the β‐hairpin structures, electron‐ or hole‐binding abilities of both termini of the β‐turn motif degenerate compared with those of the two hook structures, due to the decreased macro‐dipole polarity caused by the extending the two terminal strands. In general, the high polarity of a macro‐dipole always plays a principal role in determining charge‐relay properties through modifying the components and energies of the highest occupied and lowest unoccupied molecular orbitals of the β‐turn motif, whereas local dipoles with low polarity only play a cooperative assisting role. Further exploration is needed to identify other factors that influence relay properties in these protein motifs.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

TMBETADISC-RBF: Discrimination of beta-barrel membrane proteins using RBF networks and PSSM profiles

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. We have developed a method based on radial basis function networks and position specific scoring matrix (PSSM) profiles generated by PSI-BLAST and non-redundant protein database. Our approach with PSSM profiles has correctly predicted the OMPs with a cross-validated accuracy of 96.4% in a set of 1251 proteins, which contain 206 OMPs, 667 globular proteins and 378 alpha-helical inner membrane proteins. Furthermore, we applied our method on a dataset containing 114 OMPs, 187 TMH proteins and 195 globular proteins obtained with less than 20% sequence identity and obtained the cross-validated accuracy of 95%. This accuracy of discriminating OMPs is higher than other methods in the literature and our method could be used as an effective tool for dissecting OMPs from genomic sequences. We have developed a prediction server, TMBETADISC-RBF, which is available at http://rbf.bioinfo.tw/~sachen/OMP.html.     

Impact of Strand Number on Parallel β‐Sheet Stability

We have examined whether parallel β‐sheet secondary structure becomes more stable as the number of β‐strands increases, via comparisons among peptides designed to adopt two‐ or three‐stranded parallel β‐sheet conformations in aqueous solution. Our three‐strand design is the first experimental model of a triple‐stranded parallel β‐sheet. Analysis of the designed peptides by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy supports the hypothesis that increasing the number of β‐strands, from two to three, increases the stability of the parallel β‐sheet. We present the first experimental evidence for cooperativity in the folding of a triple‐stranded parallel β‐sheet, and we show how minimal model systems may enable experimental documentation of characteristic properties, such as CD spectra, of parallel β‐sheets.     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Antiparallel Self‐Association of a γ,α‐Hybrid Peptide: More Relevance of Weak Interactions

To learn how a preorganized peptide‐based molecular template, together with diverse weak non‐covalent interactions, leads to an effective self‐association, we investigated the conformational characteristics of a simple γ,α‐hybrid model peptide, Boc‐γ‐Abz‐Gly‐OMe. The single‐crystal X‐ray diffraction analysis revealed the existence of a fully extended β‐strand‐like structure stabilized by two non‐conventional C?H???O=C intramolecular H‐bonds. The 2D ¹H NMR ROESY experiment led us to propose that the flat topology of the urethane‐γ‐Abz‐amide moiety is predominantly preserved in a non‐polar environment. The self‐association of the energetically more favorable antiparallel β‐strand‐mimic in solid‐state engenders an unusual ‘flight of stairs’ fabricated through face‐to‐face and edge‐to‐edge Ar???Ar interactions. In conjunction with FT‐IR spectroscopic analysis in chloroform, we highlight that conformationally semi‐rigid γ‐Abz foldamer in appositely designed peptides may encourage unusual β‐strand or β‐sheet‐like self‐association and supramolecular organization stabilized via weak attractive forces.     

Design of Stable β‐Sheet‐Based Cyclic Peptide Assemblies Assisted by Metal Coordination: Selective Homo‐ and Heterodimer Formation

Metal‐directed supramolecular construction represents one of the most powerful tools to prepare a large variety of structures and functions. The ability of metals to organize different numbers and types of ligands with a variety of geometries (linear, trigonal, octahedral, etc.) expands the supramolecular synthetic architecture. We describe here the precise construction of homo‐ and heterodimeric cyclic peptide entities through coordination of a metal (Pd, Au) and to β‐sheet‐type hydrogen‐bonding interactions. The selective coordination properties of the appropriate metal allow control over the cross‐strand interaction between the two‐peptide strands.     

A HMM-based method to predict the transmembrane regions of beta-barrel membrane proteins

A novel method is developed to model and predict the transmembrane regions of beta-barrel membrane proteins. It is based on a Hidden Markov model (HMM) with architecture obeying those proteins' construction principles. The HMM is trained and tested on a non-redundant set of 11 beta-barrel membrane proteins known to date at atomic resolution with a jack-knife procedure. As a result, the method correctly locates 97% of 172 transmembrane beta-strands. Out of the 11 proteins, the barrel size for ten proteins and the overall topology for seven proteins are correctly predicted. Additionally, it successfully assigns the entire topology for two new beta-barrel membrane proteins that have no significant sequence homology to the 11 proteins. Predicted topology for two candidates for beta-barrel structure of the outer mitochondrial membrane is also presented in the paper.     

A Hybrid Ring‐Opening Metathesis Polymerization Catalyst Based on an Engineered Variant of the β‐Barrel Protein FhuA

A β‐barrel protein hybrid catalyst was prepared by covalently anchoring a Grubbs–Hoveyda type olefin metathesis catalyst at a single accessible cysteine amino acid in the barrel interior of a variant of β‐barrel transmembrane protein ferric hydroxamate uptake protein component A (FhuA). Activity of this hybrid catalyst type was demonstrated by ring‐opening metathesis polymerization of a 7‐oxanorbornene derivative in aqueous solution.     

Quantum mechanical studies on model α‐pleated sheets

Pauling and Corey proposed a pleated‐sheet configuration, now called α‐sheet, as one of the protein secondary structures in addition to α‐helix and β‐sheet. Recently, it has been suggested that α‐sheet is a common feature of amyloidogenic intermediates. We have investigated the stability of antiparallel β‐sheet and two conformations of α‐sheet in solution phase using the density functional theoretical method. The peptides are modeled as two‐strand acetyl‐(Ala)₂‐N‐methylamine. Using stages of geometry optimization and single point energy calculation at B3LYP/cc‐pVTZ//B3LYP/6‐31G* level and including zero‐point energies, thermal, and entropic contribution, we have found that β‐sheet is the most stable conformation, while the α‐sheet proposed by Pauling and Corey has 13.6 kcal/mol higher free energy than the β‐sheet. The α‐sheet that resembles the structure observed in molecular dynamics simulations of amyloidogenic proteins at low pH becomes distorted after stages of geometry optimization in solution. Whether the α‐sheets with longer chains would be increasingly favorable in water relative to the increase in internal energy of the chain needs further investigation. Different from the quantum mechanics results, AMBER parm94 force field gives small difference in solution phase energy between α‐sheet and β‐sheet. The predicted amide I IR spectra of α‐sheet shows the main band at higher frequency than β‐sheet. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

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/.     

An immunoproteomic approach for characterization of the outer membrane proteins of Salmonella Gallinarum

Salmonella enterica serovar Gallinarum (SG) is an important pathogen that causes fowl typhoid in chickens. In order to investigate SG outer membrane proteins (OMPs) as potential vaccine candidate proteins, we established a proteomic map and database of antigenic SG‐OMPs. A total of 174 spots were detected by 2DE. Twenty‐two antigen‐reactive spots were identified as nine specific proteins using PMF. OmpA was the most abundant protein among all of the identified OMPs, and it exhibited seven protein species. We conducted Western blot analysis for the SG‐OMPs in order to determine which proteins were cross‐reactive to the serovars Salmonella Enteritidis, Salmonella Typhimurium, and SG. Our results indicated that OmpA was considered to be an antigenic cross‐reactive protein among the three serovars. This study sheds new light on our understanding of cross‐protection among Salmonella serovars.     

An Antibody with a Variable‐Region Coiled‐Coil “Knob” Domain

The X‐ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent‐exposed β‐strand “stalk” fused to a disulfide crosslinked “knob” domain. We have substituted an antiparallel heterodimeric coiled‐coil motif for the β‐strand stalk in this antibody. The resulting antibody (Ab‐coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H–D exchange supports the coiled‐coil structure of the substituted peptides. Substitution of the knob‐domain of Ab‐coil with bovine granulocyte colony‐stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS‐60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled‐coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.     

The Dependence of Amyloid‐β Dynamics on Protein Force Fields and Water Models

We studied the dynamics of Aβ₄₀, involved in Alzheimer's disease, by using 21 methods combined from Amber03, Amber99sb‐ILDN, Charmm27, Charmm22*, OPLS‐2001, OPLS‐2006, OPLS‐2008, Gromos96‐43a1, Gromos96‐53a6, Gromos96‐54a7, and the water models SPC, TIP3P, TIP4P. Major differences in the structural ensembles were systematized: Amber03, Charmm27, and Gromos96‐54a7 stabilize the helices; Gromos96‐43a1 and Gromos53a6 favor the β‐strands (with Charmm22* and Amber99sb‐ILDN in between), and OPLS produces unstructured ensembles. The accuracy of the NMR chemical shifts was in the order: Charmm22*>Amber99sb‐ILDN>OPLS‐2008≈Gromos96‐43a1>Gromos96‐54a7≈OPLS‐2001>OPLS‐2006>Gromos96‐53a6>Charmm27>Amber03. The computed ³J_HNHα‐coupling constants were sensitive to experiment type and Karplus parameterization. Overall, the ensembles of Charmm22* and Amber99sb‐ILDN provided the best agreement with experimental NMR and circular dichroism data, providing a model for the real Aβ monomer ensemble. Also, the polar water model TIP3P significantly favored helix and compact conformations.     

Helical‐Ribbon Formation by a β‐Amino Acid Modified Amyloid β‐Peptide Fragment

An addition to the family : The introduction of β‐amino acid residues into a modified amyloid β peptide fragment resulted in well‐defined helical nanoribbons (see cryo‐TEM image) comprising β strands mainly oriented perpendicular to the ribbon axis. The nanoribbons order into a flow‐aligning nematic phase at higher concentration. The β‐strand nanoribbon structure is an addition to the known set of secondary structures adopted by β‐peptides.

     

1,2,3‐Triazole Bridge as Conformational Constrain in β‐Hairpin Peptides: Analysis of Hydrogen‐Bonded Positions

Conformational constrained β‐hairpin peptides are useful tool to modulate protein–protein interactions. A triazole bridge in hydrogen‐bonded positions between two antiparallel strands induces a conformational stabilization of the β‐hairpin peptide. The entity of the stability of the β‐hairpin peptide depends on the length of the bridge.     

A Molecular Dynamics Study on Controlling the Self‐Assembly of β‐Sheet Peptides with Designer Nanorings

Recently, a rational approach for constructing β‐barrel protein mimics by the self‐assembly of peptide‐based building blocks has been demonstrated. We performed molecular dynamics simulations of nanoring formation by means of the self‐assembly of designed β‐sheet‐forming peptides. Several factors contributing to the stability of the nanoring structures with respect to size were investigated. Our simulations predicted that an optimal nanoring size may be achieved by minimizing repulsions due to steric hindrance between bulky groups while maintaining favorable hydrogen‐bond interactions between neighboring β‐sheet chains. It was shown that mutations in a test peptide, in which all or half of the tryptophan residues were replaced by phenylalanine, could enable the assembly of stable nanoring structures with smaller pore sizes. Insights into the fundamental factors driving the formation of peptide‐based nanostructures are expected to facilitate the design of novel functional bionanostructures.     

Contact between the β1 and β2 Segments of α‐Synuclein that Inhibits Amyloid Formation

Conversion of the intrinsically disordered protein α‐synuclein (α‐syn) into amyloid aggregates is a key process in Parkinson’s disease. The sequence region 35–59 contains β‐strand segments β1 and β2 of α‐syn amyloid fibril models and most disease‐related mutations. β1 and β2 frequently engage in transient interactions in monomeric α‐syn. The consequences of β1–β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double‐cysteine mutant α‐synCC, with a disulfide linking β1 and β2, is aggregation‐incompetent and inhibits aggregation and toxicity of wild‐type α‐syn. We show that α‐syn delays the aggregation of amyloid‐β peptide and islet amyloid polypeptide involved in Alzheimer’s disease and type 2 diabetes, an effect enhanced in the α‐synCC mutant. Tertiary interactions in the β1–β2 region of α‐syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.