Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
... 4. CONCLUSIONS The orientation of the crystals in silver films vacuum deposited onto glass is determined by the substrate temperature and the deposition rate in the initial stages of condensation. ... Chem., 9 (1965) 70; D.Sc. Thesis,... more
... 4. CONCLUSIONS The orientation of the crystals in silver films vacuum deposited onto glass is determined by the substrate temperature and the deposition rate in the initial stages of condensation. ... Chem., 9 (1965) 70; D.Sc. Thesis, Sofia, 1967. ...
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which... more
The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3′ ends of piRNAs, bound to mature (3′ 2′ O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD of 5.0 ± 2.2 μM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues invol...
Research Interests:
Research Interests:
During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and... more
During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.
Research Interests:
Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal acquisition through anti-hemostatic... more
Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal acquisition through anti-hemostatic compounds that counteract blood clotting, platelet aggregation, vasoconstriction and host immune responses. D7 proteins are among the most abundant components of the salivary glands of several blood feeding insects. They are members of a family of proteins that have evolved through gene duplication events to encode D7 proteins of several lengths. Here, we examine the ligand binding specificity and physiological relevance of two D7 long proteins, CxD7L1 and CxD7L2, fromCulex quinquefasciatusmosquitoes, the vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host ligands involved in hemostasis, including bioactive lipids, bioge...
Research Interests:
DbpA is an DEAD-box RNA helicase implicated in RNA structural isomerization in the peptide bond formation site. In addition to the RecA-like catalytic core conserved in all of the members of DEAD-box family, DbpA contains a structured... more
DbpA is an DEAD-box RNA helicase implicated in RNA structural isomerization in the peptide bond formation site. In addition to the RecA-like catalytic core conserved in all of the members of DEAD-box family, DbpA contains a structured C-terminal domain, which is responsible for anchoring DbpA to hairpin 92 of 23S ribosomal RNA during the ribosome assembly process. Here, surface plasmon resonance was used to determine the equilibrium dissociation constant and the microscopic rate constants of the DbpA C-terminal domain association and dissociation to a fragment of 23S ribosomal RNA containing hairpin 92. Our results show that the DbpA protein's residence time on the RNA is 10 times longer than the time DbpA requires to hydrolyze one ATP. Thus, our data suggest that once bound to the intermediate ribosomal particles via its RNA-binding domain, DbpA could unwind a number of double-helix substrates before its dissociation from the ribosomal particles.
Research Interests:
The poxvirus F9 protein is a component of the vaccinia virus entry fusion complex (EFC) which consists of 11 proteins. The EFC forms a unique apparatus among viral fusion proteins and complexes. We solved the atomic structure of the F9... more
The poxvirus F9 protein is a component of the vaccinia virus entry fusion complex (EFC) which consists of 11 proteins. The EFC forms a unique apparatus among viral fusion proteins and complexes. We solved the atomic structure of the F9 ectodomain at 2.10 Å. A structural comparison to the ectodomain of the EFC protein L1 indicated a similar fold and organization, in which a bundle of five α-helices is packed against two pairs of β-strands. However, instead of the L1 myristoylation site and hydrophobic cavity, F9 possesses a protruding loop between α-helices α3 and α4 starting at Gly90. Gly90 is conserved in all poxviruses except Salmon gill poxvirus (SGPV) and Diachasmimorpha longicaudata entomopoxvirus. Phylogenetic sequence analysis of all Poxviridae F9 and L1 orthologs revealed the SGPV genome to contain the most distantly related F9 and L1 sequences compared to the vaccinia proteins studied here. The structural differences between F9 and L1 suggest functional adaptations during e...
Research Interests:
The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two... more
The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment....
Research Interests:
Research Interests:
Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. A lipocalin protein family member (LTBP1) from the saliva... more
Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. A lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, sequesters cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Crystal structures of ligand-free LTBP1 and its complexes with leukotrienes C4 (LTC4) and D4 (LTD4) reveal a conformational change during binding that brings Tyr 114 into close contact with the ligand. LTC4 is cleaved in the complex leaving free glutathione, and a C20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated. However, mutation of Tyr 114 to alanine results in a much reduced level of cleavage, as does binding of LTD4, leaving open the possibility that cleavage is protein-assisted, and may occur as part of...
Research Interests:
Research Interests:
The highly conserved H3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. Here, we present the crystal structure of H3 from vaccinia virus... more
The highly conserved H3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. Here, we present the crystal structure of H3 from vaccinia virus at 1.9Å resolution. H3 looks like a glycosyltransferase, a family of enzymes that transfer carbohydrate molecules to a variety of acceptor substrates. Like glycosyltransferases, H3 binds UDP-glucose as shown by saturation transfer difference (STD) NMR spectroscopy and this binding requires Mg(2+). Mutating the glycosyltransferase-like metal ion-binding motif in H3 greatly diminished its binding to UDP-glucose. We found by flow cytometry that H3 binds to the surface of human cells, but does not bind well to cells that are deficient in surface glycosaminoglycans. STD NMR experiments using a heparin sulfate decasaccharide confirmed that H3 binds heparin sulfate. We propose that a surface of H3 with an excess of positive charge may be the binding site ...
Research Interests:
Research Interests:
This chapter relates the intricate architecture of the L11-RNA complex to previous studies that delineated crucial features of the RNA tertiary structure and protein-RNA interface. In describing the structure, it is interesting to note... more
This chapter relates the intricate architecture of the L11-RNA complex to previous studies that delineated crucial features of the RNA tertiary structure and protein-RNA interface. In describing the structure, it is interesting to note how conservation and variation of different nucleotides and amino acids serve as a guide to critical features of the complex, and the authors use the extreme conservation of some bases to speculate about functional surfaces of the rRNA domain. Lastly, the chapter discusses the possibility that the functional role of L11-C76 is to promote a correct RNA tertiary fold. Relatively few RNA structures that have noncanonical interactions have been determined at atomic resolution, and of these only tRNA and the P4-P6 domain of group I intron have extensive tertiary structure. From nuclear magnetic resonance (NMR) studies of the free L11 RNA binding domain (L11-C76), it was known that the protein folds into three α-helices that are superimposable on the α-helices of the homeodomain class of DNA binding proteins. Covariation analysis has been an extremely powerful method for predicting rRNA secondary structure and providing clues to tertiary interactions. In melting studies of the 58-nt RNA, it was proposed that the lowest-temperature melting transition is due to unfolding of a set of tertiary interactions that link the three helical elements. In the last decade rRNA has taken center stage as the functional component of ribosomes, and it has been suggested that the primary role of ribosomal proteins is to promote RNA folding.
Research Interests:
The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born... more
The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Thermodynamics, Chemistry, Kinetics, Biophysical Chemistry, Medicine, and 15 moreBiological Sciences, Computer Simulation, Potentiometry, DIELECTRIC, Physical sciences, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Dielectric Constant, Amino Acid Substitution Rates, Recombinant Proteins, Molecule, Solvent, Protein Denaturation, Glutamic Acid, and Polarizability
The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born... more
The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.
Research Interests:
Research Interests:
Biochemistry, Chemistry, Crystallography, Electrochemistry, Magnetic Resonance Spectroscopy, and 15 moreMedicine, Glutamate, Long Range, Enzyme, Hydrogen Bond, Hydrogen Bonding, Active site, Distance Geometry, American Chemical Society, Binding Site, Biochemistry and cell biology, Energy minimization, dihedral angle, binding sites, and Medical biochemistry and metabolomics
Research Interests:
Research Interests:
Biochemistry, Chemistry, Crystallography, Catalysis, Calcium, and 12 moreMedicine, Macromolecular X-Ray Crystallography, Crystal structure, Mutation, Ligand Binding, Cooperativity, Structure activity Relationship, Ligands, Biochemistry and cell biology, DNA mutational analysis, binding sites, and Medical biochemistry and metabolomics
Research Interests:
Biochemistry, Chemistry, Crystallography, Magnetic Resonance Spectroscopy, Medicine, and 15 moreEscherichia coli, Enzyme, Protein Secondary Structure Prediction, Guanosine Triphosphate, Molecular Conformation, Protein Conformation, Amino Acid Sequence, Nucleotide, Nucleotides, Biochemistry and cell biology, Adenosine Triphosphate, Molecular Sequence Data, pyrophosphatases, binding sites, and Medical biochemistry and metabolomics
The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is... more
The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.