Biochemistry, Biophysics, and Structural Biology | Open Access Articles

Simultaneous Formation Of Functional Leading And Lagging Strand Holoenzyme Complexes On A Small, Defined Dna Substrate, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by …

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Sequence Profile Of The Parallel Β Helix In The Pectate Lyase Superfamily, Susan Heffron, Gregory R. Moe, Volker Sieber, Jerome Mengaud, Pascale Cossart, Jacqueline Vitali, Frances Jumak

Physics Faculty Publications

The parallel β helix structure found in the pectatelyasesuperfamily has been analyzed in detail. A comparative analysis of known structures has revealed a unique sequenceprofile, with a strong positional preference for specific amino acids oriented toward the interior of the parallel β helix. Using the unique sequenceprofile, search patterns have been constructed and applied to the sequence databases to identify a subset of proteins that are likely to fold into the parallel β helix. Of the 19 families identified, 39% are known to be carbohydrate-binding proteins, and 50% belong to a broad category of proteins with sequences containing leucine-rich repeats …

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The Three-Dimensional Structure Of Aspergillus Niger Pectin Lyase B At 1.7-Å Resolution1, Jacqueline Vitali, Brian Schick, Harry C.M. Kester, Jaap Visser, Frances Jurnak

Physics Faculty Publications

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel b helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel b strands, one turn of an a helix, and one turn of a 310 …

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Protein-Protein And Protein-Dna Interactions At The Bacteriophage T4 Dna Replication Fork. Characterization Of A Fluorescently Labeled Dna Polymerase Sliding Clamp, Daniel J. Sexton, Theodore E. Carver, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine …

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The Carboxyl Terminus Of The Bacteriophage T4 Dna Polymerase Is Required For Holoenzyme Complex Formation, Anthony J. Berdis, Patrice Soumillion, Stephen J. Benkovic

Chemistry Faculty Publications

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated ΔC6 exo−, is identical to wild-type exo− polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo− polymerase, the ΔC6 exo− polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo− polymerase was tested as an in vitro inhibitor of …

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Structure Of A Bovine Thrombin-Hirudin51-65 Complex Determined By A Combination Of Molecular Replacement And Graphics. Incorporation Of Known Structural Information In Molecular Replacement, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, Cris M. Olsen, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Crystals of the bovine thrombin-hirudins_51-65 complex have space group P6₁22 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in …

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Crystallization Of Hemoglobins Ii And Iii Of The Symbiont-Harboring Clam Lucina Pectinata, M. A. Doyle, Jacqueline Vitali, J. B. Wittenberg, S. N. Vinogradov, D. A. Walz, B. F.P. Edwards, P. D. Martin

Physics Faculty Publications

Diffraction data to 2.7 A resolution were measured on crystals of the homotetramers of components II and III of the cytoplacmic hemoglobin of the symbiont-harboring clam Lucina pectinata. Even though the crystallization conditions are different and the sequence homology of the two hemoglobins is only 63%, the crystals are isomorphous to each other and to the heterotetramer Hb II/III, implying that the residues primarily involved in the intermolecular interactions and responsible for crystal cohesion may be invariant.

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Dissection Of An Antibody-Catalyzed Reaction, Jon D. Stewart, Joseph F. Krebs, Gary Siuzdak, Anthony J. Berdis, David B. Smithrud, Stephen J. Benkovic

Chemistry Faculty Publications

Antibody 43C9 accelerates the hydrolysis of a p-nitroanilide by a factor of 2.5 x 10(5) over the background rate in addition to catalyzing the hydrolysis of a series of aromatic esters. Since this represents one of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mechanism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetrahedral intermediate that forms upon nucleophilic attack by hydroxide on the amide substrate. Further studies, however, revealed that the catalytic mechanism is more complex and involves …

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The 2′-Phosphate Of Nadp Is Critical For Optimum Productive Binding To 6-Phosphogluconate Dehydrogenase From Candida Utilis, Anthony J. Berdis, Paul F. Cook

Chemistry Faculty Publications

Initial velocity studies obtained with alternative dinucleotide substrates for the 6-phosphogluconate dehydrogenase reaction suggest that the 2′-phosphate is critical for the optimum productive binding of the dinucleotide substrate. Initial velocity patterns obtained by varying 6-phosphogluconate at different fixed levels of NAD are nearly parallel with apparent competitive substrate inhibition by 6-phosphogluconate at pH 7 and below but intersect to the left of the ordinate at pH 8 and above. Dead-end inhibition studies indicate that the mechanism is random at all pH values. Data are interpreted in terms of a random mechanism with marked antagonism in the binding of NAD and …

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Site-Directed Mutagenesis By Double Polymerase Chain Reaction: Megaprimer Method, Sailen Barik

Biological, Geological, and Environmental Faculty Publications

The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A and B represent the "flanking" primers that can map either within the cloned gene or outside the gene (i.e., within the vector sequence) and M represents the internal "mutant" primer containing the desired base change. …

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The Structure Of A Complex Of Bovine &-Thrombin And Recombinant Hirudin At 2.8-A Resolution, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, William D. Robertson, Jerome B. Lazar, Richard C. Winant, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in …

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Using Xenon As A Heavy Atom For Determining Phases In Sperm Whale Metmyoglobin, Jacqueline Vitali, Arthur H. Robbins, Steven C. Almo, Robert F. Tilton

Physics Faculty Publications

Xenon gas can be used as a heavy atom for determining phases in a protein. We demonstrate that an interpretable electron density map can be obtained for sperm whale metmyoglobin from a single xenon derivative using iterative single isomorphous replacement with the anomalous scattering method.

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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Simultaneous Formation Of Functional Leading And Lagging Strand Holoenzyme Complexes On A Small, Defined Dna Substrate, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

Sequence Profile Of The Parallel Β Helix In The Pectate Lyase Superfamily, Susan Heffron, Gregory R. Moe, Volker Sieber, Jerome Mengaud, Pascale Cossart, Jacqueline Vitali, Frances Jumak

Physics Faculty Publications

The Three-Dimensional Structure Of Aspergillus Niger Pectin Lyase B At 1.7-Å Resolution1, Jacqueline Vitali, Brian Schick, Harry C.M. Kester, Jaap Visser, Frances Jurnak

Physics Faculty Publications

Protein-Protein And Protein-Dna Interactions At The Bacteriophage T4 Dna Replication Fork. Characterization Of A Fluorescently Labeled Dna Polymerase Sliding Clamp, Daniel J. Sexton, Theodore E. Carver, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

The Carboxyl Terminus Of The Bacteriophage T4 Dna Polymerase Is Required For Holoenzyme Complex Formation, Anthony J. Berdis, Patrice Soumillion, Stephen J. Benkovic

Chemistry Faculty Publications

Structure Of A Bovine Thrombin-Hirudin51-65 Complex Determined By A Combination Of Molecular Replacement And Graphics. Incorporation Of Known Structural Information In Molecular Replacement, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, Cris M. Olsen, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Crystallization Of Hemoglobins Ii And Iii Of The Symbiont-Harboring Clam Lucina Pectinata, M. A. Doyle, Jacqueline Vitali, J. B. Wittenberg, S. N. Vinogradov, D. A. Walz, B. F.P. Edwards, P. D. Martin

Physics Faculty Publications

Dissection Of An Antibody-Catalyzed Reaction, Jon D. Stewart, Joseph F. Krebs, Gary Siuzdak, Anthony J. Berdis, David B. Smithrud, Stephen J. Benkovic

Chemistry Faculty Publications

The 2′-Phosphate Of Nadp Is Critical For Optimum Productive Binding To 6-Phosphogluconate Dehydrogenase From Candida Utilis, Anthony J. Berdis, Paul F. Cook

Chemistry Faculty Publications

Site-Directed Mutagenesis By Double Polymerase Chain Reaction: Megaprimer Method, Sailen Barik

Biological, Geological, and Environmental Faculty Publications

The Structure Of A Complex Of Bovine &-Thrombin And Recombinant Hirudin At 2.8-A Resolution, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, William D. Robertson, Jerome B. Lazar, Richard C. Winant, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Using Xenon As A Heavy Atom For Determining Phases In Sperm Whale Metmyoglobin, Jacqueline Vitali, Arthur H. Robbins, Steven C. Almo, Robert F. Tilton

Physics Faculty Publications