T. Palzkill - Academia.edu (original) (raw)

Papers by T. Palzkill

Research paper thumbnail of Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime

Journal of Biological Chemistry, 1994

Recently, TEM p-lactamase variants with amino acid substitutions in the active-site pocket of the... more Recently, TEM p-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238SE240K double mutant. Each enzyme was purified to homogeneity and * This work was supported by National Institutes of Health Grant part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research paper thumbnail of Roles of Amino Acids 161 to 179 in the PSE-4 Ω Loop in Substrate Specificity and in Resistance to Ceftazidime

Antimicrobial Agents and Chemotherapy, 1998

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against... more The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Ω loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Ω loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

Research paper thumbnail of Characterization of a PSE-4 Mutant with Different Properties in Relation to Penicillanic Acid Sulfones: Importance of Residues 216 to 218 in Class A β-Lactamases

Antimicrobial Agents and Chemotherapy, 1998

Class A β-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and ... more Class A β-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 β-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in theKm from 68 μM for the wild type to 271 μM for the mutant for carbenicillin and 33 to 216 μM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases inKi values were observed for the mutant e...

Research paper thumbnail of Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase

Journal of Bacteriology, 1996

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. T... more Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also i...

Research paper thumbnail of Selection of functional signal peptide cleavage sites from a library of random sequences

Journal of Bacteriology, 1994

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-... more The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position f...

Research paper thumbnail of Identification of human single-chain antibodies with broad reactivity for noroviruses

Protein engineering, design & selection : PEDS, 2014

Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose no... more Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose norovirus infections are needed. The goal of this study was to identify antibodies that have broad reactivity of binding to various genogroups of norovirus. A human scFv phage display library was used to identify two antibodies, HJT-R3-A9 and HJT-R3-F7, which bind to both genogroups I and II norovirus virus-like particles (VLPs). Mapping experiments indicated that the HJT-R3-A9 clone binds to the S-domain while the HJT-R3-F7 clone binds the P-domain of the VP1 capsid protein. In addition, a family of scFv antibodies was identified by elution of phage libraries from the GII.4 VLP target using a carbohydrate that serves as an attachment factor for norovirus on human cells. These antibodies were also found to recognize both GI and GII VLPs in enzyme-linked immunosorbent assay (ELISA) experiments. The HJT-R3-A9, HJT-R3-F7 and scFv antibodies identified with carbohydrate elution were shown to d...

Research paper thumbnail of Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

Journal of Virology, 2014

Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viropl... more Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close pr...

Research paper thumbnail of Epitope mapping and use of epitope-specific antisera to characterize the VP5⁎ binding site in rotavirus SA11 NSP4

Virology, 2008

Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural prot... more Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural protein 4 (NSP4), the first characterized viral enterotoxin, is a 28-kDa glycoprotein that has pleiotropic functions in RV infection and pathogenesis. NSP4 has multiple forms enabling it to perform its different functions. Dissecting such functions could be facilitated by use of epitope-specific antibodies. This work mapped the epitopes for the monoclonal antibody B4-2/55 and three polyclonal antisera generated against synthetic SA11 NSP4 peptides corresponding to residues 114-135, 120-147, and 150-175. The epitope for B4-2/55 mapped to residues 100-118, wherein residues E105, R108 and E111 are critical for antibody binding. Antiserum generated to two peptides (aa114-135 and aa120-147) with enterotoxin activity each recognize a single but distinct epitope. The epitope for the peptide antiserum to aa114-135 was mapped to residues 114-125 with highly conserved residues T117/T118, E120, and E122 being critical for antibody binding. The peptide antiserum to aa120-147 binds to NSP4 at residues 130-140 and residues Q137-T138 are critical for this epitope. Finally, the epitope for the antiserum to peptide aa150-175 mapped to residues 155-170, wherein residues E160 and E170 are critical for antibody binding. Knowledge of the binding sites of domain-specific antibodies can aid in further characterizing different functions of NSP4. To demonstrate this, we characterized the interaction between NSP4 and VP5 ⁎ [K D = 0.47 μM] and show that binding of NSP4 to VP5 ⁎ is blocked by antibody to NSP4 aa114-135 and aa120-147, but not aa150-175. The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.

Research paper thumbnail of A broad-spectrum peptide inhibitor of  -lactamase identified using phage display and peptide arrays

Protein Engineering Design and Selection, 2003

Hydrolysis of b-lactam antibiotics by b-lactamase enzymes is the most common mechanism of bacteri... more Hydrolysis of b-lactam antibiotics by b-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of b-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A b-lactamases. Here we utilized phage display to identify peptides that bind to the class A b-lactamase, TEM-1. The binding af®nity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 b-lactamase with a K i of 136 mM. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 b-lactamase with a K i of 42 mM and the class C b-lactamase, P99, with a K i of 140 mM, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-b-lactam, broad-spectrum peptidomimetic inhibitors of b-lactamases.

Research paper thumbnail of Susceptibility of  -lactamase to core amino acid substitutions

Protein Engineering Design and Selection, 1999

To determine which amino acids in TEM-1 β-lactamase are important for its structure and function,... more To determine which amino acids in TEM-1 β-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 β-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A β-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other β-lactamases. It is possible that position 76 varies in the gene family because β-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the β-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A β-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Research paper thumbnail of Identification and characterization of  -lactamase inhibitor protein-II (BLIP-II) interactions with  -lactamases using phage display

Protein Engineering Design and Selection, 2010

Research paper thumbnail of Structure-function analysis of  -helix H4 using PSE-4 as a model enzyme representative of class A  -lactamases

Protein Engineering Design and Selection, 2000

We extracted maximum information for structure-function analysis of the PSE-4 class A β-lactamase... more We extracted maximum information for structure-function analysis of the PSE-4 class A β-lactamase by random replacement mutagenesis of three contiguous codons in the H4 α-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structurefunction studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 α-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face π-π interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 α-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 α-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 α-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Research paper thumbnail of Protein minimization by random fragmentation and selection

Protein Engineering Design and Selection, 2001

Protein-protein interactions are involved in most biological processes and are important targets ... more Protein-protein interactions are involved in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 β-lactamase (K i ⍧ 0.1 nM). To aid in the development of new inhibitors of β-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 β-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 β-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit β-lactamase.

Research paper thumbnail of A secondary drug resistance mutation of TEM-1 beta -lactamase that suppresses misfolding and aggregation

Proceedings of the National Academy of Sciences, 2000

Research paper thumbnail of Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis

PLoS ONE, 2010

In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA ... more In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters.

Research paper thumbnail of Design of Potent beta -Lactamase Inhibitors by Phage Display of beta -Lactamase Inhibitory Protein

Journal of Biological Chemistry, 2000

Research paper thumbnail of Structural Insight into the Kinetics and  Cp of Interactions between TEM-1  -Lactamase and  -Lactamase Inhibitory Protein (BLIP)

Journal of Biological Chemistry, 2008

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in ␤-lactamase i... more In a previous study, we examined thermodynamic parameters for 20 alanine mutants in ␤-lactamase inhibitory protein (BLIP) for binding to TEM-1 ␤-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 ␤-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (⌬G ‫؍‬ ϳ؊13 kcal mol ؊1 and ⌬Cp ‫؍‬ ϳ؊0.8 kcal mol ؊1 K ؊1) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (⌬G ‫؍‬ ϳ؊8.5 kcal mol ؊1 and ⌬Cp ‫؍‬ ϳ؊0.27 kcal mol ؊1 K ؊1). We previously determined that BLIP Tyr 51 is a canonical and Trp 150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp 49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp 49 of W150A moves more than 4 Å to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp 150 to alanine substitution, and also reveals a strong long distance coupling between Trp 150 and Asp 49 of BLIP, because these two residues are more than 25 Å apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.

Research paper thumbnail of Cephalosporin Substrate Specificity Determinants of TEM-1 beta -Lactamase

Journal of Biological Chemistry, 1997

Research paper thumbnail of Analysis of the Binding Forces Driving the Tight Interactions between β-Lactamase Inhibitory Protein-II (BLIP-II) and Class A β-Lactamases

Journal of Biological Chemistry, 2011

3 The abbreviations used are: BLIP, ␤-lactamase inhibitory protein; ITC, isothermal titration cal... more 3 The abbreviations used are: BLIP, ␤-lactamase inhibitory protein; ITC, isothermal titration calorimetry; ACES, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid.

Research paper thumbnail of BAC Library of T. pallidum DNA in E. coli

Genome Research, 2002

Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large in... more Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidumchromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of th...

Research paper thumbnail of Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime

Journal of Biological Chemistry, 1994

Recently, TEM p-lactamase variants with amino acid substitutions in the active-site pocket of the... more Recently, TEM p-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238SE240K double mutant. Each enzyme was purified to homogeneity and * This work was supported by National Institutes of Health Grant part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research paper thumbnail of Roles of Amino Acids 161 to 179 in the PSE-4 Ω Loop in Substrate Specificity and in Resistance to Ceftazidime

Antimicrobial Agents and Chemotherapy, 1998

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against... more The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Ω loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Ω loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

Research paper thumbnail of Characterization of a PSE-4 Mutant with Different Properties in Relation to Penicillanic Acid Sulfones: Importance of Residues 216 to 218 in Class A β-Lactamases

Antimicrobial Agents and Chemotherapy, 1998

Class A β-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and ... more Class A β-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 β-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in theKm from 68 μM for the wild type to 271 μM for the mutant for carbenicillin and 33 to 216 μM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases inKi values were observed for the mutant e...

Research paper thumbnail of Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase

Journal of Bacteriology, 1996

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. T... more Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also i...

Research paper thumbnail of Selection of functional signal peptide cleavage sites from a library of random sequences

Journal of Bacteriology, 1994

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-... more The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position f...

Research paper thumbnail of Identification of human single-chain antibodies with broad reactivity for noroviruses

Protein engineering, design & selection : PEDS, 2014

Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose no... more Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose norovirus infections are needed. The goal of this study was to identify antibodies that have broad reactivity of binding to various genogroups of norovirus. A human scFv phage display library was used to identify two antibodies, HJT-R3-A9 and HJT-R3-F7, which bind to both genogroups I and II norovirus virus-like particles (VLPs). Mapping experiments indicated that the HJT-R3-A9 clone binds to the S-domain while the HJT-R3-F7 clone binds the P-domain of the VP1 capsid protein. In addition, a family of scFv antibodies was identified by elution of phage libraries from the GII.4 VLP target using a carbohydrate that serves as an attachment factor for norovirus on human cells. These antibodies were also found to recognize both GI and GII VLPs in enzyme-linked immunosorbent assay (ELISA) experiments. The HJT-R3-A9, HJT-R3-F7 and scFv antibodies identified with carbohydrate elution were shown to d...

Research paper thumbnail of Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

Journal of Virology, 2014

Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viropl... more Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close pr...

Research paper thumbnail of Epitope mapping and use of epitope-specific antisera to characterize the VP5⁎ binding site in rotavirus SA11 NSP4

Virology, 2008

Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural prot... more Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural protein 4 (NSP4), the first characterized viral enterotoxin, is a 28-kDa glycoprotein that has pleiotropic functions in RV infection and pathogenesis. NSP4 has multiple forms enabling it to perform its different functions. Dissecting such functions could be facilitated by use of epitope-specific antibodies. This work mapped the epitopes for the monoclonal antibody B4-2/55 and three polyclonal antisera generated against synthetic SA11 NSP4 peptides corresponding to residues 114-135, 120-147, and 150-175. The epitope for B4-2/55 mapped to residues 100-118, wherein residues E105, R108 and E111 are critical for antibody binding. Antiserum generated to two peptides (aa114-135 and aa120-147) with enterotoxin activity each recognize a single but distinct epitope. The epitope for the peptide antiserum to aa114-135 was mapped to residues 114-125 with highly conserved residues T117/T118, E120, and E122 being critical for antibody binding. The peptide antiserum to aa120-147 binds to NSP4 at residues 130-140 and residues Q137-T138 are critical for this epitope. Finally, the epitope for the antiserum to peptide aa150-175 mapped to residues 155-170, wherein residues E160 and E170 are critical for antibody binding. Knowledge of the binding sites of domain-specific antibodies can aid in further characterizing different functions of NSP4. To demonstrate this, we characterized the interaction between NSP4 and VP5 ⁎ [K D = 0.47 μM] and show that binding of NSP4 to VP5 ⁎ is blocked by antibody to NSP4 aa114-135 and aa120-147, but not aa150-175. The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.

Research paper thumbnail of A broad-spectrum peptide inhibitor of  -lactamase identified using phage display and peptide arrays

Protein Engineering Design and Selection, 2003

Hydrolysis of b-lactam antibiotics by b-lactamase enzymes is the most common mechanism of bacteri... more Hydrolysis of b-lactam antibiotics by b-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of b-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A b-lactamases. Here we utilized phage display to identify peptides that bind to the class A b-lactamase, TEM-1. The binding af®nity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 b-lactamase with a K i of 136 mM. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 b-lactamase with a K i of 42 mM and the class C b-lactamase, P99, with a K i of 140 mM, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-b-lactam, broad-spectrum peptidomimetic inhibitors of b-lactamases.

Research paper thumbnail of Susceptibility of  -lactamase to core amino acid substitutions

Protein Engineering Design and Selection, 1999

To determine which amino acids in TEM-1 β-lactamase are important for its structure and function,... more To determine which amino acids in TEM-1 β-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 β-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A β-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other β-lactamases. It is possible that position 76 varies in the gene family because β-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the β-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A β-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Research paper thumbnail of Identification and characterization of  -lactamase inhibitor protein-II (BLIP-II) interactions with  -lactamases using phage display

Protein Engineering Design and Selection, 2010

Research paper thumbnail of Structure-function analysis of  -helix H4 using PSE-4 as a model enzyme representative of class A  -lactamases

Protein Engineering Design and Selection, 2000

We extracted maximum information for structure-function analysis of the PSE-4 class A β-lactamase... more We extracted maximum information for structure-function analysis of the PSE-4 class A β-lactamase by random replacement mutagenesis of three contiguous codons in the H4 α-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structurefunction studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 α-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face π-π interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 α-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 α-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 α-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Research paper thumbnail of Protein minimization by random fragmentation and selection

Protein Engineering Design and Selection, 2001

Protein-protein interactions are involved in most biological processes and are important targets ... more Protein-protein interactions are involved in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 β-lactamase (K i ⍧ 0.1 nM). To aid in the development of new inhibitors of β-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 β-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 β-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit β-lactamase.

Research paper thumbnail of A secondary drug resistance mutation of TEM-1 beta -lactamase that suppresses misfolding and aggregation

Proceedings of the National Academy of Sciences, 2000

Research paper thumbnail of Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis

PLoS ONE, 2010

In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA ... more In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters.

Research paper thumbnail of Design of Potent beta -Lactamase Inhibitors by Phage Display of beta -Lactamase Inhibitory Protein

Journal of Biological Chemistry, 2000

Research paper thumbnail of Structural Insight into the Kinetics and  Cp of Interactions between TEM-1  -Lactamase and  -Lactamase Inhibitory Protein (BLIP)

Journal of Biological Chemistry, 2008

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in ␤-lactamase i... more In a previous study, we examined thermodynamic parameters for 20 alanine mutants in ␤-lactamase inhibitory protein (BLIP) for binding to TEM-1 ␤-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 ␤-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (⌬G ‫؍‬ ϳ؊13 kcal mol ؊1 and ⌬Cp ‫؍‬ ϳ؊0.8 kcal mol ؊1 K ؊1) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (⌬G ‫؍‬ ϳ؊8.5 kcal mol ؊1 and ⌬Cp ‫؍‬ ϳ؊0.27 kcal mol ؊1 K ؊1). We previously determined that BLIP Tyr 51 is a canonical and Trp 150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp 49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp 49 of W150A moves more than 4 Å to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp 150 to alanine substitution, and also reveals a strong long distance coupling between Trp 150 and Asp 49 of BLIP, because these two residues are more than 25 Å apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.

Research paper thumbnail of Cephalosporin Substrate Specificity Determinants of TEM-1 beta -Lactamase

Journal of Biological Chemistry, 1997

Research paper thumbnail of Analysis of the Binding Forces Driving the Tight Interactions between β-Lactamase Inhibitory Protein-II (BLIP-II) and Class A β-Lactamases

Journal of Biological Chemistry, 2011

3 The abbreviations used are: BLIP, ␤-lactamase inhibitory protein; ITC, isothermal titration cal... more 3 The abbreviations used are: BLIP, ␤-lactamase inhibitory protein; ITC, isothermal titration calorimetry; ACES, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid.

Research paper thumbnail of BAC Library of T. pallidum DNA in E. coli

Genome Research, 2002

Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large in... more Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidumchromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of th...