Molecular models for the core components of the flagellar type-III secretion complex

We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments

Algorithms for incorporating prior topological information in HMMs: application to transmembrane proteins

BACKGROUND: Hidden Markov Models (HMMs) have been extensively used in computational molecular biology, for modelling protein and nucleic acid sequences. In many applications, such as transmembrane protein topology prediction, the incorporation of limited amount of information regarding the topology, arising from biochemical experiments, has been proved a very useful strategy that increased remarkably the performance of even the top-scoring methods. However, no clear and formal explanation of the algorithms that retains the probabilistic interpretation of the models has been presented so far in the literature. RESULTS: We present here, a simple method that allows incorporation of prior topological information concerning the sequences at hand, while at the same time the HMMs retain their full probabilistic interpretation in terms of conditional probabilities. We present modifications to the standard Forward and Backward algorithms of HMMs and we also show explicitly, how reliable predictions may arise by these modifications, using all the algorithms currently available for decoding HMMs. A similar procedure may be used in the training procedure, aiming at optimizing the labels of the HMM's classes, especially in cases such as transmembrane proteins where the labels of the membrane-spanning segments are inherently misplaced. We present an application of this approach developing a method to predict the transmembrane regions of alpha-helical membrane proteins, trained on crystallographically solved data. We show that this method compares well against already established algorithms presented in the literature, and it is extremely useful in practical applications. CONCLUSION: The algorithms presented here, are easily implemented in any kind of a Hidden Markov Model, whereas the prediction method (HMM-TM) is freely available for academic users at , offering the most advanced decoding options currently available

Prediction of β-barrel membrane proteins by searching for restricted domains

BACKGROUND: The identification of beta-barrel membrane proteins out of a genomic/proteomic background is one of the rapidly developing fields in bioinformatics. Our main goal is the prediction of such proteins in genome/proteome wide analyses. RESULTS: For the prediction of beta-barrel membrane proteins within prokaryotic proteomes a set of parameters was developed. We have focused on a procedure with a low false positive rate beside a procedure with lowest false prediction rate to obtain a high certainty for the predicted sequences. We demonstrate that the discrimination between beta-barrel membrane proteins and other proteins is improved by analyzing a length limited region. The developed set of parameters is applied to the proteome of E. coli and the results are compared to four other described procedures. CONCLUSION: Analyzing the beta-barrel membrane proteins revealed the presence of a defined membrane inserted beta-barrel region. This information can now be used to refine other prediction programs as well. So far, all tested programs fail to predict outer membrane proteins in the proteome of the prokaryote E. coli with high reliability. However, the reliability of the prediction is improved significantly by a combinatory approach of several programs. The consequences and usability of the developed scores are discussed

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency

Structural organization of gap junction channels

AbstractGap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge

Influence of Single and Multiple Histidine Residues and their Ionization Properties on Transmembrane Helix Dynamics, Orientations and Fraying

Since aromatic and charged residues are often present in various locations of transmembrane helices of integral membrane proteins, their impacts on the molecular properties of transmembrane proteins and their interactions with lipids are of particular interest in many studies. In this work, I used solid-state deuterium NMR spectroscopy in designed model peptide GWALP23 [GGALW(LA)6LWLAGA] with selective deuterium labels to addresses the pH dependence and influence of single and multiple “guest” histidine residues in the orientation and dynamic behaviors of transmembrane proteins. The mutations include Gly to His (G2/22 to H2/22), Trp to His (W5/19 to H5/19) and Leu to His (L8/16 to H8/16). For the glycine to histidine substitutions, either one or both, the peptides show similar biophysical properties to the host GWALP23 peptide, with modest motional averaging and tilted transmembrane orientations that scale with bilayer thicknesses. Yet, the dynamic motion about the average azimuthal rotation increases significantly when the helix carries only H22. However, when the tryptophan residues, W5 and/or W19 are replaced by histidines, the new histidine residues effectively anchor the transmembrane α-helix, providing similar transmembrane topology. A consistent ~30° shift in helix rotation is observed for Trp to His substitutions and found to be terminal-dependent. Modifying the core sequence of GWALP23 with His residues at positions 8 and 16 provides some interesting insights. The peptide is significantly tilted in DLPC, has multiple orientations in DMPC and surface bound in DPoPC and DOPC lipid bilayers, where the bilayer thicknesses increase consecutively from DLPC to DOPC. Further analysis for peptide with only H8 was performed. Results indicate multiple signal resonances, similar to -H8,16, but in a thicker lipid bilayer. Moreover, the helix with H8 alone significantly responds with pH in DLPC and DMPC lipids and two titration points for H8 was calculated. Finally, mutation of GWALP23 with two adjacent histidines at the N-terminal end (positions 4 and 5) causes a large increase in the motional averaging about helix azimuthal rotation, which in turn obscures the actual orientation and the peptide is found to adopt a very small tilt angle

The Integrin Equilibrium: Balancing Protein-Protein and Protein-Lipid interactions

On circulating platelets, the integrin fibrinogen receptor &alphaIIb&beta3 favors inactive conformations. Platelets rapidly activate &alphaIIb&beta3 to bind fibrinogen, mediating a platelet clot. Resting &alphaIIb&beta3 is stabilized by interactions between the &alphaIIb and &beta3 transmembrane domains. Binding of talin-1 and kindlin-3 to the integrin cytoplasmic domain stabilizes separation of the TMs and receptor activation. Src family kinases are needed for transmission of extracellular signals into the cell. We have sought to better understand how signals are transmitted across the &alphaIIb&beta3 TM domain. First we characterized the structure and dynamics of the active and inactive integrin cytoplasmic domain to determine how motifs that bind talin-1 and kindlin-3 are affected by the integrin activation state and the membrane environment. The &alphaIIb&beta3 cytoplasmic domain is disorded, while the &beta3 subunit contains two &alpha-helices, which interact with the phospholipid bilayer. The close proximity of &alphaIIb to &beta3 in the inactive state induces a kink that projects the &beta-chain parallel to the membrane surface. This kink is likely to stabilize interactions between helices in &beta3 with the membrane and possibly compete with binding to talin-1 and kindlin-3. The &beta3 cytoplasmic domain becomes increasingly dynamic further from the membrane, suggesting that distal kindlin-3 binding residues should be more accessible to interact with their binding partners. We also studied the interaction between talin-1 and the &beta3 cytoplasmic domain to determine how the membrane environment affects formation of the talin-1/&beta3 complex. The membrane significantly increases the affinity of talin-1 for &beta3, but that the ternary integrin/talin-1/membrane complex is not significantly more stable than the talin-1/membrane complex alone. We also performed preliminary experiments characterizing the interaction between &alphaIIb&beta3 and both kindlin-3 and c-Src. Unlike talin-1, kindlin-3 does not require the membrane to bind the integrin cytoplasmic domain with high affinity. The interaction between c-Src and the integrin was extremely weak suggesting that it might require colocalization through other interactions. Finally, we developed a simplified model showing how thermodynamic coupling between integrin subunits might allow &alphaIIb&beta3 to adopt multiple activation states

Structural characterization of helices A and B of Kv7.2 channel bound to Calmodulin. The calcium effect

217 p.Los canales neuronales heterodiméricos Kv7.2 y Kv7.3 son el sustrato molecular de la corriente depotasio no-inactivante dominada ¿Corriente M¿. Ésta juega un papel crítico en el control de laexcitabilidad neuronal. Su mal funcionamiento desencadena consecuencias fatales causantes deenfermedades como la epilepsia neonatal benigna (BFNC) que pueden estar asociadas a encefalopatias yretraso mental. La calmodulina es una proteína auxiliar sensible al calcio que es esencial para el correctoensamblaje, tráfico y función de dichos canales. Mientras que la compresión de la regulación de estoscanales ha alcanzado niveles razonables debido a estudios bioquímicos y electrofisiológicos, poco se sabesobre la estructura y conformación. El propósito de la presente tesis es el aumentar los conocimientosestructurales de los canales Kv7.2 con la meta de mejorar el entendimiento de cómo se da la regulaciónpor calcio de estos canales, y a su vez mejorar el conocimiento de los mecanismos por los que ciertaspatologías ocurren. Para ello la tesis presenta tres fases: 1) Mejora de la expresión recombinante del Cterminaldel canal, que se ha conseguido gracias a la coexpresion de la calmodulina y de la region Cterminaldel canal. 2) Caracterización estructural del complejo CaM/Hélices_AB mediante RMN, dondese observa cómo la calmodulina abraza dos helices del C-terminal (A y B) que están orientadas de maneraantiparalela. 3) Estudio del impacto del calcio sobre el complejo donde se demuestra que no se dancambios drásticos grandes en el complejo, con lo que se puede deducir que la señalización por calcio hade ser un mecanismo más sutil, posiblemente mediante cambios en las dinámicas locales o mediantemecanismos alostéricos