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PubMed2002

Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR.

Bacterial sigma factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the -10 and -35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of sigma is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the -35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a sigma70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.

Amino Acid Sequence↗
PubMed1992

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.

Amino Acid Sequence↗
PubMed2004

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces.

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.

Amino Acid Sequence↗
PubMed1982

Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry.

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single-cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Since large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.

Alpharetrovirus↗
PubMed1996

Finding and visualizing nucleic acid base stacking.

Base stacking is one of the primary factors stabilizing nucleic acid structure. Yet, methods for locating stacking interactions in DNA and RNA are rare and methods for displaying stacking are rarer still. We present here simple, automated procedures to search nucleic acid molecules for base-base and base-oxygen stacking and to display these interactions graphically in a manner that readily conveys both the location and the quality of the interaction. The method makes no a priori assumptions about relative base positions when searching for stacking, nor does it rely on empirical energy functions. This is a distinct advantage for two reasons. First, the relative contributions of the forces stabilizing stacked bases are unknown. Second, the electrostatic and hydrophobic components of base stacking are both poorly defined by existing potential energy functions.

Algorithms↗
PubMed2006

Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems.

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties.

DNA, Bacterial↗
PubMed2005

Stability of intracellular influenza virus nucleocapsid protein oligomers.

Stability of A/Duck/Ukrainae/63 (H3N8) influenza virus intracellular NP oligomers was studied using reducing agents, denaturants, detergents, salts, various pH and a range of temperatures. The results obtained indicate that influenza virus NP oligomers are noncovalently stabilized, and NP subunits are not linked by disulfide bonds. NP oligomers are thermostable and SDS resistant. Urea and high ionic strength also do not dissociate avian influenza virus intracellular NP oligomers. However, NP oligomers are completely dissociated at pH < 5. The data obtained suggest that hydrophobic bonds together with the electrostatic interactions take part in the stabilization of compact conformation of influenza virus NP oligomers. It was also shown that intrachain disulfides revealed in nascent NPs are reduced in NP subunits of NP oligomers, and this probably contributes to the stability and compactness of the oligomers.

Detergents↗
PubMed1987

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin.

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Amino Acid Sequence↗
PubMed2006

Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1.

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.

Amino Acid Sequence↗
PubMed1999

Nephelometric determination of micro amounts of nucleic acids with protamine sulfate.

Nucleic acids can form large particle complexes with protamine sulfate by electrostatic forces, which results in strong light scattering. Based on this, a nephelometric method is described for sensitive and convenient determination of nucleic acids with protamine sulfate by using a common spectrofluorimeter. Maximum light scattering is produced in the range of pH 2.2-4.4 with the same excitation and emission wavelengths at 365 nm. Under optimal conditions, the calibration curves are linear in the range 0.05-60.0 micrograms cm-3 for nucleic acids. The corresponding detection limits are 12.5 ng cm-3 for calf thymus DNA, 9.0 ng cm-3 for fish sperm DNA, and 18.0 ng cm-3 for yeast RNA, respectively. Six synthetic samples are determined with satisfactory results. The relative standard deviation of five replicate measurements is 3.2% for 2.0 micrograms cm-3 calf thymus DNA.

Animals↗
PubMed2004

An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair.

The human CCR4-NOT complex is a global regulator of RNA polymerase II transcription. Recently, we showed that the RING domain CNOT4 subunit contains intrinsic ubiquitin-protein ligase (E3) activity. Here we show that binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective. To understand the basis for this interaction, we identified several basic residues of UbcH5B important for binding to CNOT4 by mutational analysis. Subsequently, we tested pairs of UbcH5B and CNOT4 mutants for restoration of interaction. Concomitant charge-alteration of E49 of CNOT4 and K63 of UbcH5B restored binding and re-created a functional enzyme pair, indicative of an electrostatic interaction between these residues. The corresponding amino acids in the yeast orthologues can also be used to create a similarly designed E2-E3 enzyme pair. These are the first examples of altered-specificity E2-E3 enzyme pairs and give further insight into how E2-E3 specificity is obtained.

Amino Acid Substitution↗
PubMed1983

Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3.

We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.

Amino Acid Sequence↗
PubMed2007

A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate.

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.

Acylation↗
PubMed1998

Simulations of the molecular dynamics of nucleic acids.

The growing amount of high quality molecular dynamics simulations generated using the latest methodological developments and force fields has led to a sharper understanding of the forces underlying the dynamics of biomolecular systems, as well as to stimulating insights into the structure and catalysis of nucleic acids. It is now clear that inclusion of long-range electrostatic interactions and of the aqueous and ionic environment is necessary for producing realistic and accurate simulations. Yet, many papers hint at a force field and protocol dependence of the results and thus contain the seeds for the future improvements that will be necessary for deepening our understanding of recognition phenomena and folding of nucleic acids.

Crystallization↗
PubMed2003

Binding interactions between the core central domain of 16S rRNA and the ribosomal protein S15 determined by molecular dynamics simulations.

The goal of the current study is to utilize molecular dynamic (MD) simulations to investigate the dynamic behavior of 16S rRNA in the presence and absence of S15 and to identify the binding interactions between these two molecules. The simulations show that: (i) 16S rRNA remains in a highly folded structure when it is bound to S15; (ii) in the absence of S15, 16S rRNA significantly alters its conformation and transiently forms conformations that are similar to the bound structure that make it available for binding with S15; (iii) the unbound rRNA spends the majority of its time in extended conformations. The formation of the extended conformations is a result of the molecule reaching a lower electrostatic energy and the formation of the highly folded, crystal-like conformation is a result of achieving a lower solvation energy. In addition, our MD simulations show that 16S rRNA and S15 bind across the major groove of helix 22 (H22) via electrostatic interactions. The negatively charged phosphate groups of G658, U740, G741 and G742 bind to the positively charged S15 residues Lys7, Arg34 and Arg37. The current study provides a dynamic view of the binding of 16S rRNA with S15.

Base Sequence↗
PubMed1977

On the binding of Mg2+ and Mn2+ to tRNA.

In this paper previous binding studies of Mg2+ and Mn2+ ions to tRNA's are reconsidered. Binding data of some representative examples are interpreted including interactions between charges located on the macroion. Both curved and bell-shaped Scatchard plots can be accounted for quantitatively if corrections are made for electrostatic interactions and, if necessary, for the effect of conformational changes on these interactions. It appears that there is no need to invoke more than one class of binding sites on tRNA's, meaning that the experimental binding data can be described using the same intrinsic pK value for all phosphate groups.

Binding Sites↗
PubMed2000

Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies.

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non-competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg(2+) ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg(2+) for common binding sites on RNase P holoenzyme.

Animals↗