Presence Of Transmembrane Helix Research Articles

In-Depth Annotation of the Drosophila Bithorax-Complex Reveals the Presence of Several Alternative ORFs That Could Encode for Motif-Rich Peptides.

It is recognized that a large proportion of eukaryotic RNAs and proteins is not produced from conventional genes but from short and alternative (alt) open reading frames (ORFs) that are not captured by gene prediction programs. Here we present an in silico prediction of altORFs by applying several selecting filters based on evolutionary conservation and annotations of previously characterized altORF peptides. Our work was performed in the Bithorax-complex (BX-C), which was one of the first genomic regions described to contain long non-coding RNAs in Drosophila. We showed that several altORFs could be predicted from coding and non-coding sequences of BX-C. In addition, the selected altORFs encode for proteins that contain several interesting molecular features, such as the presence of transmembrane helices or a general propensity to be rich in short interaction motifs. Of particular interest, one altORF encodes for a protein that contains a peptide sequence found in specific isoforms of two Drosophila Hox proteins. Our work thus suggests that several altORF proteins could be produced from a particular genomic region known for its critical role during Drosophila embryonic development. The molecular signatures of these altORF proteins further suggests that several of them could make numerous protein–protein interactions and be of functional importance in vivo.

Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis.

To clone, express and purify 2-methylcitrate synthase (Rv1131) gene of Mycobacterium tuberculosis (M. tuberculosis) and to study its structural characteristics using various bioinformatics tools. Rv1131 gene was amplified by polymerase chain reaction using M. tuberculosis H37Rv genomic DNA and cloned into pGEM-T easy vector and sequenced. The gene was sub-cloned in pET28c vector, expressed in Escherichia coli BL21 (E. coli BL21) (DE3) cells and the recombinant protein was identified by Western blotting. The protein was purified using Nickel affinity chromatography and the structural characteristics like sub-cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools. Tertiary structure of the protein and phylogenetic analysis was also established by in silico analysis. The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction. In silico analysis showed that the protein contains no signal peptide and transmembrane helices. Active site prediction showed that the protein has histidine and aspartic acid residues at 242, 281 & 332 positions respectively. Phylogenetic analysis showed 100% homology with major mycobacterial species. Secondary structure predicts 2-methylcitrate synthase contain 51.9% alpha-helix, 8.7% extended strand and 39.4% random coils. Tertiary structure of the protein was also established. The enzyme 2-methylcitrate synthase from M. tuberculosis H37Rv has been successfully expressed and purified. The purified protein will further be utilized to develop assay methods for screening new inhibitors.

Transmembrane helix 6 observed at the interface of β2AR homodimers in blind docking studies

Peptide– and protein–protein dockings were carried out on β2-adrenergic receptor (β2AR) to confirm the presence of transmembrane helix 6 (TM6) at the interface region between two β2AR monomers, thereby its possible role in dimerization as suggested in numerous experimental and computational studies. Initially, a portion of TM6 was modeled as a peptide consisting of 23 residues and blindly docked to β2AR monomer using a rigid body approach. Interestingly, all highest score conformations preferred to be near TM5 and TM6 regions of the receptor. Furthermore, longer peptides generated from a whole TM region were blindly docked to β2AR using the same rigid body approach. This yielded a total of seven docked peptides, each derived from one TM helix. Most interestingly, for each peptide, TM6 was among the most preferred binding site region in the receptor. Besides the peptide dockings, two β2AR monomers were blindly docked to each other using a full rigid-body search of docking orientations, which yielded a total of 16,000 dimer conformations. Each dimer was then filtered according to a fitness value based on the membrane topology. Among 149 complexes that met the topology requirements, 102 conformers were composed of two monomers oriented in opposite directions, whereas in the remaining 47, the monomers were arranged in parallel. Lastly, all 149 conformers were clustered based on a root mean-squared distance value of 6 Å. In agreement with the peptide results, the clustering yielded the largest population of conformers with the highest Z-score value having TM6 at the interface region.

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

An MFS Transporter-Like ORF from MDRAcinetobacter baumanniiAIIMS 7 Is Associated with Adherence and Biofilm Formation on Biotic/Abiotic Surface

A major facilitator superfamily (MFS) transporter-like open reading frame (ORF) of 453 bp was identified in a pathogenic strain Acinetobacter baumannii AIIMS 7, and its association with adherence and biofilm formation was investigated. Reverse transcription PCR (RT-PCR) showed differential expression in surface-attached biofilm cells than nonadherent cells. In vitro translation showed synthesis of a ~17 kDa protein, further confirmed by cloning and heterologous expression in E. coli DH5α. Up to 2.1-, 3.1-, and 4.1- fold biofilm augmentation was observed on abiotic (polystyrene) and biotic (S. cerevisiae/HeLa) surface, respectively. Scanning electron microscopy (SEM) and gfp-tagged fluorescence microscopy revealed increased adherence to abiotic (glass) and biotic (S. cerevisiae) surface. Extracellular DNA(eDNA) was found significantly during active growth; due to probable involvement of the protein in DNA export, strong sequence homology with MFS transporter proteins, and presence of transmembrane helices. In summary, our findings show that the putative MFS transporter-like ORF (pmt) is associated with adherence, biofilm formation, and probable eDNA release in A. baumannii AIIMS 7.

Molecular Simulations of Lipid Flip-Flop in the Presence of Model Transmembrane Helices

The transport of lipids between membrane leaflets, also known as flip-flop, is a key process in regulating the lipid composition of biological membranes. It is also important for the growth of biogenic membranes that are the site for lipid synthesis. It has been shown that the mere presence of transmembrane alpha-helical peptides or proteins enhances the rate lipid flip-flop [Kol et al. (2001) Biochemistry 40, 10500-10506]. Using computational models of natural phospholipids with different headgroups, we calculated the free energy profiles for transferring single phospholipids from bulk water to the center of a dioleylphosphatidylcholine (DOPC) bilayer in the presence of transmembrane helices. The free energy barrier for phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) flip-flop decreased by a few kilojoules per mole when a WALP23 or KALP23 peptide was present in the membrane, while the barrier for PC was not affected. We observed large bilayer deformations during lipid flip-flop when the hydrophilic headgroup is in the hydrophobic interior of the bilayer. The presence of KALP23 or WALP23 decreased the size and stability of these defects, suggesting integral membrane proteins affect the mechanism of flip-flop. There was a large decrease in the free energy of desorption for PE and PG when transmembrane peptides were present. This suggests specific PE and PG interactions with the peptide have a large affect on their stability in the membrane, with implications on cellular lipid and protein trafficking.

Evolution of Prokaryotic Two-Component System Signaling Pathways: Gene Fusions and Fissions

Two-component systems (TCSs) are common signal transduction systems, typically comprising paired histidine protein kinase (HK) and response regulator (RR) proteins. In many examples, it appears RR and HK genes have fused, producing a "hybrid kinase " We have characterized a set of prokaryotic genes encoding RRs, HKs, and hybrid kinases, enabling characterization of gene fusion and fission. Primary factors correlating with fusion rates are the presence of transmembrane helices in HKs and the presence of DNA-binding domains in RRs, features that require correct (and separate) spatial location. In the absence of such features, there is a relative abundance of fused genes. The order of paired HK and RR genes and the nucleotide distance between encoded domains also correlate with apparent gene fusion rates. We propose that localization requirements and relative positioning of encoded domains within TCS genes affect the function (and therefore retention) of hybrid kinases resulting from gene fusion.

Oligomerization, Membrane Anchoring, and Cellulose-binding Characteristics of AbpS, a Receptor-like Streptomyces Protein

Streptomyces reticuli produces a 34.6-kDa surface-anchored protein (AbpS) whose surface-exposed N terminus binds strongly to Avicel, a dominantly crystalline type of cellulose. The generation of a large set of mutated abpS-genes and the subsequent analysis of the corresponding proteins in vitro as well as in vivo in a Streptomyces host allow the assignment of the following characteristics for AbpS. (i) Amino acid residues participating directly in the cellulose-interaction are located at the N terminus. (ii) As ascertained by cross-linking experiments, AbpS forms homotetramers in its soluble as well as cellulose-bound form. (iii) The intermolecular assembly of four AbpS molecules is governed by two domains (including amino acids 60-110 and 161-212). Both domains possess large portions of alpha-helical regions in which hydrophobic amino acids are located on one side as known from coiled-coil proteins. (iv) The C-terminal part of AbpS comprising 35 amino acids contains a transmembrane domain. Due to the surface-exposed N terminus of AbpS and the presence of transmembrane helix the C terminus has to be situated in the cytoplasm of the S. reticuli hyphae. Thus AbpS connects the interior of the mycelia with the extracellular space and binds cellulose using a unique cellulose-binding module.

NORSp: Predictions of long regions without regular secondary structure.

Many structurally flexible regions play important roles in biological processes. It has been shown that extended loopy regions are very abundant in the protein universe and that they have been conserved through evolution. Here, we present NORSp, a publicly available predictor for disordered regions in protein. Specifically, NORSp predicts long regions with NO Regular Secondary structure. Upon user submission of a protein sequence, NORSp will analyse the protein for its secondary structure, presence of transmembrane helices and coiled-coil. It will then return email to the user about the presence and position of disordered regions. NORSp can be accessed from http://cubic.bioc.columbia.edu/services/NORSp/.