Structure of the Cytoplasmic Loop between Putative Helices II and III of the Mannitol Permease of Escherichia coli: A Tryptophan and 5-Fluorotryptophan Spectroscopy Study

In this work, four single tryptophan (Trp) mutants of the dimeric mannitol transporter of Escherichia coli, EIImtl, are characterized using Trp and 5-fluoroTrp (5-FTrp) fluorescence spectroscopy. The four positions, 97, 114, 126, and 133, are located in a region shown by recent studies to be involved in the mannitol translocation process. To spectroscopically distinguish between the Trp positions in each subunit of dimeric EIImtl, 5-FTrp was biosynthetically incorporated because of its much simpler photophysics compared to those of Trp. The steady-state and time-resolved fluorescence methodologies used point out that all four positions are in structured environments, both in the absence and in the presence of a saturating concentration of mannitol. The fluorescence decay of all 5-FTrp-containing mutants was highly homogeneous, suggesting similar microenvironments for both probes per dimer. However, Stern-Volmer quenching experiments using potassium iodide indicate different solvent accessibilities for the two probes at positions 97 and 133. A 5 Å two-dimensional (2D) projection map of the membrane-embedded IICmtl dimer showing 2-fold symmetry is available. The results of this work are in better agreement with a 7 Å projection map from a single 2D crystal on which no symmetry was imposed.

?2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ?2-microglobulin (?2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ?2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ?2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ?2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ?2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ?2m amyloid-associated osteoarticular tissue destruction in DRA

Latherin: A Surfactant Protein of Horse Sweat and Saliva

Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg ml−1), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 Å thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material

Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism

Lactate oxidase (LOx), recognized to selectively catalyze the lactate oxidation in complex matrices, has been highlighted as preferable biorecognition element for the development of lactate biosensors. In a previous work, we have demonstrated that LOx crosslinking on a modified screen-printed electrode results in a dual range lactate biosensor, with one of the analysis linear range (4 to 50 mM) compatible with lactate sweat levels. It was advanced that such behavior results from an atypical substrate inhibition process. To understand such inhibition phenomena, this work relies in the study of LOx structure when submitted to increased substrate concentrations. The results found by fluorescence spectroscopy and dynamic light scattering of LOx solutions, evidenced conformational changes of the enzyme, occurring in presence of inhibitory substrate concentrations. Therefore, the inhibition behavior found at the biosensor, is an outcome of LOx structural alterations as result of a pH-dependent mechanism promoted at high substrate concentrations.Spanish Ministry of Science and Innovation (MICINN), Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (FEDER) (TEC20013-40561-P and MUSSEL RTC-2015-4077-2). Hugo Cunha-Silva would like to acknowledge funding from the Spanish Ministry of Economy (BES-2014-068214

Mechanism of Assembly of the Dimanganese-Tyrosyl Radical Cofactor of Class Ib Ribonucleotide Reductase: Enzymatic Generation of Superoxide Is Required for Tyrosine Oxidation via a Mn(III)Mn(IV) Intermediate

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The Fe[superscript III][subscript 2]-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe[superscript II][subscript 2]-NrdB, O[subscript 2], and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn[superscript III][subscript 2]-Y• cofactor in their NrdF subunit. Mn[superscript II][subscript 2]-NrdF does not react with O[subscript 2], but it binds the reduced form of a conserved flavodoxin-like protein, NrdI[subscript hq], which, in the presence of O[subscript 2], reacts to form the Mn[superscript III][subscript 2]-Y• cofactor. Here we investigate the mechanism of assembly of the Mn[superscript III][subscript 2]-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn[superscript II][subscript 2]-NrdF, NrdI[subscript hq], and O[subscript 2] has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI[subscript hq] reduces O[subscript 2] to O[subscript 2]•– (40–48 s[superscript –1], 0.6 mM O[subscript 2]), the O[subscript 2]•– channels to and reacts with Mn[superscript II][subscript 2]-NrdF to form a Mn[superscript III]Mn[superscript IV] intermediate (2.2 ± 0.4 s[superscript –1]), and the Mn[superscript III]Mn[superscript IV] species oxidizes tyrosine to Y• (0.08–0.15 s[superscript –1]). Controlled production of O[subscript 2]•– by NrdI[subscript hq] during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn[superscript II][subscript 2] cluster with O[subscript 2] and satisfies the requirement for an “extra” reducing equivalent in Y• generation.National Institutes of Health (U.S.) (Grant GM81393)United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Biophysical Characterization and Membrane Interaction of the Two Fusion Loops of Glycoprotein B from Herpes Simplex Type I Virus

The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV) requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes

Staphylococcal phenotypes induced by naturally occurring and synthetic membrane-interactive polyphenolic β-lactam resistance modifiers.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation

Does the partial molar volume of a solute reflect the free energy of hydrophobic solvation?

Halogenated heterocyclic ligands are widely used as the potent and frequently selective inhibitors of protein kinases. However, the exact contribution of the hydrophobic solvation of a free ligand is rarely accounted for the balance of interactions contributing to the free energy of ligand binding. Herein, we propose a new experimental method based on volumetric data to estimate the hydrophobicity of a ligand. We have tested this approach for a series of ten variously halogenated benzotriazoles, the binding affinity of which to the target protein kinase CK2 was assessed with the use of thermal shift assay. According to the hierarchical clustering procedure, the excess volume, defined as the difference between the experimentally determined partial molar volume and the calculated in silico molecular volume, was found to be distant from any commonly used hydrophobicity descriptors of the ligand. The excess volume, however, properly predicts solute binding affinity. On the way, we have proved that the binding of halogenated benzotriazoles to the protein kinase CK2 is driven mostly by hydrophobic interaction