Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier

BACKGROUND: Membrane proteins are estimated to represent about 25% of open reading frames in fully sequenced genomes. However, the experimental study of proteins remains difficult. Considerable efforts have thus been made to develop prediction methods. Most of these were conceived to detect transmembrane helices in polytopic proteins. Alternatively, a membrane protein can be monotopic and anchored via an amphipathic helix inserted in a parallel way to the membrane interface, so-called in-plane membrane (IPM) anchors. This type of membrane anchor is still poorly understood and no suitable prediction method is currently available. RESULTS: We report here the "AmphipaSeeK" method developed to predict IPM anchors. It uses a set of 21 reported examples of IPM anchored proteins. The method is based on a pattern recognition Support Vector Machine with a dedicated kernel. CONCLUSION: AmphipaSeeK was shown to be highly specific, in contrast with classically used methods (e.g. hydrophobic moment). Additionally, it has been able to retrieve IPM anchors in naively tested sets of transmembrane proteins (e.g. PagP). AmphipaSeek and the list of the 21 IPM anchored proteins is available on NPS@, our protein sequence analysis server

NmPin from the marine thaumarchaeote Nitrosopumilus maritimus is an active membrane associated prolyl isomerase.

We cordially thank Alma Rute for excellent technical assistance and the DFG (GRK 1431-1 and 1431-2) for financial support (PB). We thank the Microscope and Histology Facility of the University of Aberdeen for providing their equipment. We also thank Jacob Hargreaves for proofreading the manuscript.Peer reviewedPublisher PD

Investigation into the membrane interactive properties of the escherichia coli low molecular weight penicillin-binding proteins

Various results have suggested that in Escherichia coli murein assembly may involve a protein complex(es) which could include low molecular mass penicillin-binding prpteins (PBPs). These proteins include PBP4, PBP5 and PBP6 which are penicillin sensitive enzymes associated with the periplasmic face of the inter membrane. The levels of these associations have been linked to enzymic activity and elucidation of the mechanism(s) involved in these associations may help identify and understand the regulation of this putative protein complex. It is currently accepted that the membrane associations of PBP5 and PBPÔ involve C-terminal amphiphilic cz-helices and such helices are ubiquitously employed in the lipid associations of membrane interactive protein molecules. Whether such helical structure features in the membrane associations of PBP4 or indeed if this protein is membrane bound or soluble, are, as yet, open questions. The focus of this research has been to investigate the lipid and membrane interactions of PBP4, PBP5 and PBP6 and in particular, to investigate the role played by these interactions of the C-terminal region of these proteins. Haemolytic analysis has shown that peptide homologues of the PBP5 and PBP6 C-terminal regions, P5 and P6, are active at the membrane interface and CD analysis has shown that these peptides possess a capacity for a-helix formation. CD and pressure - area isotherm analysis of monolayers formed from PS and P6 have shown that these peptides are able to adopt a-helical structure at an air - water interface. Monolayer studies have shown that P5 and P6 are able to interact with lipids and that these interactions are characterised by minor requirements for anionic lipid and the involvement of predominantly hydrophobic forces which are enhanced by low pH. Similar characteristics were revealed when perturbant washes and Western blotting were used to investigate the interactions of PBP5 with membranes derived from a mutant E. coli strain, HDL 11, in which the level of anionic lipid can be controlled. Overall, these results strongly support the hypothesis that C-terminal amphiphilic a-helices feature in PBP5 and PBP6 membrane anchoring. Molecular area determinations have implied that a peptide homologue of the PBP4 C-terminal region, P4 is able to adopt a-helical structure and this was confirmed by CD analysis. P4 showed no haemolytic activity but the peptide was found to interact generally with lipid monolayers. These monolayer interactions were characterised by a requirement for anionic lipid and involved predominantly electrostatic forces, which were enhanced by low pH. Similar characteristics but with no detectable requirement for anionic lipid were revealed when perturbant washes and chemiluminesence were used to investigate the interactions of PBP4 with membranes of the overproducing strain HB 10 I/pBK4 and those of HDL1 1. It is suggested that the PBP4 C-terminal region may play a role in PBP4 - membrane anchoring. Using chemiluminesence, no soluble form of PBP4 could be detected in the wild type E. coli, MRE600, suggesting that in wild type strains, PBP4 is exclusively membrane bound. It is suggested that PBP4 - membrane anchoring occurs at a specific binding site and overall, may differ fundamentally from that of PBP5 and PBP6

Assessing the local structural quality of transmembrane protein models using statistical potentials (QMEANBrane)

Motivation: Membrane proteins are an important class of biological macromolecules involved in many cellular key processes including signalling and transport. They account for one third of genes in the human genome and >50% of current drug targets. Despite their importance, experimental structural data are sparse, resulting in high expectations for computational modelling tools to help fill this gap. However, as many empirical methods have been trained on experimental structural data, which is biased towards soluble globular proteins, their accuracy for transmembrane proteins is often limited. Results: We developed a local model quality estimation method for membrane proteins (‘QMEANBrane') by combining statistical potentials trained on membrane protein structures with a per-residue weighting scheme. The increasing number of available experimental membrane protein structures allowed us to train membrane-specific statistical potentials that approach statistical saturation. We show that reliable local quality estimation of membrane protein models is possible, thereby extending local quality estimation to these biologically relevant molecules. Availability and implementation: Source code and datasets are available on request. Contact: [email protected] Supplementary Information: Supplementary data are available at Bioinformatics onlin

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

<p/> <p>Background</p> <p>Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues.</p> <p>Results</p> <p>We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information.</p> <p>Conclusion</p> <p>For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments.</p> <p>Reviewers</p> <p>This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian.</p

Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We have performed extensive molecular dynamics simulations of these five PrP mutants and compared their dynamics and conformations to wild-type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases