Structural basis and synergism of ATP and Na activation in bacterial K uptake system KtrAB.

The K uptake system KtrAB is essential for bacterial survival in low K environments. The activity of KtrAB is regulated by nucleotides and Na. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP.

The Antiviral Activity of Varenicline against Dengue Virus Replication during the Post-Entry Stage.

Dengue virus (DENV) poses a significant global health challenge, with millions of cases each year. Developing effective antiviral drugs against DENV remains a major hurdle. Varenicline is a medication used to aid smoking cessation, with anti-inflammatory and antioxidant effects.

Molecular chaperone TRiC governs avian reovirus replication by protecting outer-capsid protein σC and inner core protein σA and non-structural protein σNS from ubiquitin- proteasome degradation.

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of gene (8) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield.

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBT.

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na as the driving force. The crystal structures of two bacterial homologues ASBT and ASBT have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na and substrate in the conformational isomerization remains unclear.

Inhibition of the clinical isolates of Acinetobacter baumannii by Pseudomonas aeruginosa: In vitro assessment of a case-based study.

Background: The global rise in nosocomial infections associated with gram-negative bacteria and the spread of multi-drug resistant Acinetobacter baumannii (MDR-AB) pose public health concerns. This study investigates the inhibitory effects and possible inhibitory mechanism of Pseudomonas aeruginosa (PA) on selected clinical strains of A. baumannii (AB) isolated from Taiwanese patients.

Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants.

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids.

Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins.

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis.

Crystallographic analysis of the Staphylococcus epidermidis lipase involved in esterification in aqueous solution.

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303-Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.

Parity-dependent hairpin configurations of repetitive DNA sequence promote slippage associated with DNA expansion.

Repetitive DNA sequences are ubiquitous in life, and changes in the number of repeats often have various physiological and pathological implications. DNA repeats are capable of interchanging between different noncanonical and canonical conformations in a dynamic fashion, causing configurational slippage that often leads to repeat expansion associated with neurological diseases. In this report, we used single-molecule spectroscopy together with biophysical analyses to demonstrate the parity-dependent hairpin structural polymorphism of TGGAA repeat DNA.

Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel-Chromomycin Complex Resulting in Large-Scale DNA Deformation.

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. Ni (Chro) (Chro=chromomycin A3) binds specifically to the minor groove of (CCG) repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the Ni chelate itself.

A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence.

DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA.

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals.

Crystal structure of vespid phospholipase A(1) reveals insights into the mechanism for cause of membrane dysfunction.

Vespid phospholipase A1 (vPLA1) from the black-bellied hornet (Vespa basalis) catalyzes the hydrolysis of emulsified phospholipids and shows potent hemolytic activity that is responsible for its lethal effect. To investigate the mechanism of vPLA1 towards its function such as hemolysis and emulsification, we isolated vPLA1 from V. basalis venom and determined its crystal structure at 2.

Structure-based virtual screening and experimental validation of the discovery of inhibitors targeted towards the human coronavirus nucleocapsid protein.

Nucleocapsid protein (NP), an essential RNA-binding viral protein in human coronavirus (CoV)-infected cells, is required for the replication and transcription of viral RNA. Recent studies suggested that human CoV NP is a valid target for antiviral drug development. Based on this aspect, structure-based virtual screening targeting nucleocapsid protein (NP) was performed to identify good chemical starting points for medicinal chemistry.

Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK.

Cyclic di-AMP (c-di-AMP) is a relatively new member of the family of bacterial cyclic dinucleotide second messengers. It has attracted significant attention in recent years because of the abundant roles it plays in a variety of Gram-positive bacteria. The structural features that allow diverse bacterial proteins to bind c-di-AMP are not fully understood.

Structure determination of an integral membrane protein at room temperature from crystals in situ.

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software.

GFP-based expression screening of membrane proteins in insect cells using the baculovirus system.

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins.

SDAR: a practical tool for graphical analysis of two-dimensional data.

Background: Two-dimensional data needs to be processed and analysed in almost any experimental laboratory. Some tasks in this context may be performed with generic software such as spreadsheet programs which are available ubiquitously, others may require more specialised software that requires paid licences. Additionally, more complex software packages typically require more time by the individual user to understand and operate.

Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms.

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target.

Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.

High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2).

Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures.

The crystal structure of calcium-bound annexin Gh1 from Gossypium hirsutum and its implications for membrane binding mechanisms of plant annexins.

Plant annexins show distinct differences in comparison with their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins. The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.