Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in .

The genome of contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K and or Na for protons across the membrane. Based on phenotypic analysis of chromosomal , , and triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K/H antiporters and might play a role in the acid tolerance response of as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues.

Solution structure of the cytoplasmic domain of NhaP2 a K/H antiporter from Vibrio cholera.

NhaP2 is a K/H antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker.

Solution structure and oligomeric state of the E. coliglycerol facilitator.

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium.

Physiological, Structural, and Functional Analysis of the Paralogous Cation-Proton Antiporters of NhaP Type from .

The transmembrane K/H antiporters of NhaP type of (Vc-NhaP1, 2, and 3) are critical for maintenance of K homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of at low pHs suggesting their possible role in the acid tolerance response (ATR) of . Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting and other NhaP-expressing pathogens.

Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.

Background: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay.

The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales.

Folding and stability of the aquaglyceroporin GlpF: Implications for human aqua(glycero)porin diseases.

Aquaporins are highly selective polytopic transmembrane channel proteins that facilitate the permeation of water across cellular membranes in a large diversity of organisms. Defects in aquaporin function are associated with common diseases, such as nephrogenic diabetes insipidus, congenital cataract and certain types of cancer. In general, aquaporins have a highly conserved structure; from prokaryotes to humans.

The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis.

Structure-activity relationships in ultrashort cationic lipopeptides: the effects of amino acid ring constraint on antibacterial activity.

Taking a minimalistic approach in efforts to lower the cost for the development of new synthetic antimicrobial peptides, ultrashort cationic lipopeptides were designed to mimic the amphiphilic nature crucial for their activity but with only a very short peptide sequence ligated to a lipidic acid. Nine ultrashort cationic lipopeptides were prepared to study the effects of ring constraint in the amino acid side chain of the peptide component. USCL-PCat1, consisting of only four L-4R-aminoproline residues and acylated with palmitic acid at the N-terminus, was found to populate a polyproline II helical secondary conformation that is stable to different pHs and temperatures using circular dichroism.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin.

Conformational dynamics of a membrane transport protein probed by H/D exchange and covalent labeling: the glycerol facilitator.

Glycerol facilitator (GF) is a tetrameric membrane protein responsible for the selective permeation of glycerol and water. Each of the four GF subunits forms a transmembrane channel. Every subunit consists of six helices that completely span the lipid bilayer, as well as two half-helices (TM7 and TM3).

Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein-calmodulin interactions.

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat).

Intrinsic disorder and function of the HIV-1 Tat protein.

The type 1 Human Immunodeficiency Virus transcriptional regulator Tat is a small RNA-binding protein essential for viral gene expression and replication. The protein binds to a large number of proteins within infected cells and non-infected cells, and has been demonstrated to impact a wide variety of cellular activities. Early circular dichroism studies showed a lack of regular secondary structure in the protein whereas proton NMR studies suggested several different conformations.

High yield expression and purification of HIV-1 Tat1-72 for structural studies.

The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes.

Alamethicin topology in phospholipid membranes by oriented solid-state NMR and EPR spectroscopies: a comparison.

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition.

Urea as a protein destabilizing agent in electrospray ionisation.

Urea is well known as a denaturant of proteins, but there is also evidence that millimolar amounts of urea may in fact stabilize protein complexes. Advances in mass spectrometric analysis have given us the opportunity to test the effect of urea on several noncovalent complexes in buffered solutions. We expected to see lower charge states if folded proteins were more compact (and therefore more stable), and higher charge states if the proteins were denatured.

The influence of sterols on the conformation of recombinant mitochondrial porin in detergent.

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood.

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy.

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy.

Stability of the glycerol facilitator in detergent solutions.

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence.

Two-step folding of recombinant mitochondrial porin in detergent.

Precise information regarding the transmembrane topology of mitochondrial porin is essential for understanding the mechanisms by which this protein functions. Porin acts as a channel in the outer membrane and interacts with small solutes and proteins to regulate mitochondrial function. The acquisition of high-resolution structural data requires a method of maintaining high concentrations of unaggregated, properly folded porin.

HIV-1 Tat is a natively unfolded protein: the solution conformation and dynamics of reduced HIV-1 Tat-(1-72) by NMR spectroscopy.

Tat (transactivator of transcription) is a small RNA-binding protein that plays a central role in the regulation of human immunodeficiency virus type 1 replication and in approaches to treating latently infected cells. Its interactions with a wide variety of both intracellular and extracellular molecules is well documented. A molecular understanding of the multitude of Tat activities requires a determination of its structure and interactions with cellular and viral partners.