Sampling of structure and sequence space of small protein folds.

Nature only samples a small fraction of the sequence space that can fold into stable proteins. Furthermore, small structural variations in a single fold, sometimes only a few amino acids, can define a protein's molecular function. Hence, to design proteins with novel functionalities, such as molecular recognition, methods to control and sample shape diversity are necessary.

Carbene-Stabilized Disilicon as a Silicon-Transfer Agent: Synthesis of a Dianionic Silicon Tris(dithiolene) Complex.

Reaction of carbene-stabilized disilicon (1) with the lithium-based dithiolene radical (2 ) affords the first dianionic silicon tris(dithiolene) complex (3). Notably, the formation of 3 represents the unprecedented utilization of carbene-stabilized disilicon (1) as a silicon-transfer agent. The nature of 3 was probed by multinuclear NMR spectroscopy, single-crystal X-ray diffraction, and DFT computations.

Direct Sodium Channel Regulation by Calmodulin.

Many aspects of the sophisticated mechanism of sodium channel regulation by Ca and calmodulin remain unresolved and controversial. In this issue of Structure, Johnson et al. (2018) provide compelling structural and functional evidence clarifying considerably how calmodulin engages the inactivation gate of the sodium channel and the consequences for regulation.

Glucose consumption in carbohydrate mixtures by phosphotransferase-system mutants of Escherichia coli.

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted.

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth.

Solution structure and properties of AlgH from Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria.

Accumulation of d-glucose from pentoses by metabolically engineered Escherichia coli.

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E.

Membranous adenylyl cyclase 1 activation is regulated by oxidation of N- and C-terminal methionine residues in calmodulin.

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress.

Proton-induced reactivity of NO⁻ from a {CoNO}⁸ complex.

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO(-)), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors.

Synergistic and inhibitory effects of aminopeptidase peptides on Bacillus thuringiensis Cry11Ba toxicity in the mosquito Anopheles gambiae.

Cry11Ba produced by Bacillus thuringiensis subsp. jegathesan is an active toxin for larvae of the mosquito Anopheles gambiae. A 106-kDa aminopeptidase N (APN), called AgAPN2, was previously identified as a Cry11Ba receptor in A.

Determinants of affinity and activity of the anti-sigma factor AsiA.

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation.

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110.

Cloning, high yield overexpression, purification, and characterization of AlgH, a regulator of alginate biosynthesis in Pseudomonas aeruginosa.

The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate.

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin.

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes.

Design of a calcium-binding protein with desired structure in a cell adhesion molecule.

Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties. To design de novo Ca2+-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca2+-induced conformational change.

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin.

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA.

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation.

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels.

Expression, purification, and structural study of the EC4 domain of E-cadherin.

The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides. The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E. coli.

Characterization of the interactions between the bacteriophage T4 AsiA protein and RNA polymerase.

The anti-sigma factor AsiA effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the sigma(70) subunit, denoted 4.1 and 4.2.

House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding.

We report that cytochrome b(5) (cyt b(5)) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b(5) that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180 degrees rotation about the alpha-gamma-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.

Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress.

We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines.

Oxidation of Met144 and Met145 in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase.

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM.