V. J . Davisson | Purdue University (original) (raw)

Papers by V. J . Davisson

Research paper thumbnail of NMR structure analysis of the DNA nine base pair duplex D(CATGAGTAC) D(GTAC(NP3)CATG)

Research paper thumbnail of Prediction of drug toxicity in HepG2 cells using laser scanning cytometry

Toxicology Letters, 2010

(24) and 335-192 (47)]; CLZ [(m/z 327-270 (25) and 327-192 (44)]; NCLZ [(m/z 313-270 (25) and 313... more (24) and 335-192 (47)]; CLZ [(m/z 327-270 (25) and 327-192 (44)]; NCLZ [(m/z 313-270 (25) and 313-192 (41)]. Mobile phase constituted of 50% methanol with 0.1% formic acid. The flow rate is 0.6 mL/min. Total analysis time is 1.5 min. A group of 200 clinical plasma samples were analyzed and results compared with LC-MS/MS methods. Limit of detection was 1 ng/mL for CLZ and NCLZ. Limit of quantification was 5 ng/mL. The within run precision and accuracy for QC samples at 150, 300 and 600 ng/mL were between 3.69-10.11% and −0.01% to −10.31%, respectively. The correlation (r 2) between the FIA-MS/MS procedure and LC-MS/MS procedure was greater than 0.98. The FIA-MS/MS procedure was capable of analyzing CLZ and NCLZ in human plasma samples efficiently and accurately.

Research paper thumbnail of Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Nucleosides, Nucleotides & Nucleic Acids, 2005

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2&am... more Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

Research paper thumbnail of Mechanism-based inactivation of Escherichia coli .beta.-hydroxydecanoyl thiol ester dehydrase: assignment of the imidazole nitrogen-15 NMR resonances and determination of the structure of the alkylated histidine

Journal of the American Chemical Society, 1993

Nitrogen-1 5 nuclear magnetic resonance spectroscopy was used to determine the structure of the a... more Nitrogen-1 5 nuclear magnetic resonance spectroscopy was used to determine the structure of the active-site histidine-70 adduct formed when @-hydroxydecanoyl thiol ester dehydrase from Escherichia coli reacts with the mechanism-based inactivator S-(3-decynoyl)-N-acetylcysteamine (3-decynoyl-NAC). In order to obtain the amount of labeled enzyme necessary for spectral studies, thefabA gene, which encodes dehydrase, was overexpressed to give dehydrase as 1520% of soluble protein. To simplify the interpretation of the NMR spectra, the non-active-site histidine residue His-129 was converted to an asparagine residue using site-directed mutagenesis. The specific activity and response of the mutant to 3-decynoyl-NAC are unaltered. It is known that type @ ('pyridine-like") nitrogens in imidazoles resonate 60-80 ppm downfield of type a ('pyrrole-like") nitrogens. To assign the imidazole nitrogen resonances in dehydrase, wild-type and mutant enzymes were labeled with I5NH4Cl or with ['5N6']histidine. Analysis of the I5N NMR spectra allowed the assignment of the resonances of the imidazole nitrogens of His-129 and His-70. For His-70, the spectra show that N*' resonates upfield of Nf2 in the native enzyme and is therefore a type a nitrogen. In the inactivated enzyme the signals are reversed, and Nf2 is a type a nitrogen. These results demonstrate that Nf2 of His-70 becomes alkylated upon inactivation of dehydrase with 3-decynoyl-NAC and thus is the probable locus of active-site basicity in the normal reactions catalyzed by dehydrase. In addition, the imidazole nitrogen chemical shifts suggest that N*' may be involved in a hydrogen bond in native dehydrase but that Nf2 is not. The mechanistic implications of this are discussed. (I) Bloch, K. In The Enzymes, 3rd 4.

Research paper thumbnail of Stereochemical course of the Escherichia coli imidazole glycerol phosphate dehydratase reaction

Journal of the American Chemical Society, 1993

The va,st majority of enzyme-catalyzed dehydrations involve suhstretes wit,h the departing hydrog... more The va,st majority of enzyme-catalyzed dehydrations involve suhstretes wit,h the departing hydrogen and hydroxyl group sit.iiated a and 6, respectively, to a carbonyl or an imine group.' (2 5) Hansen, P. E. In Annual Reports on N M R Spectroscopy; Webb, G.

Research paper thumbnail of The Raman detection of peptide tyrosine phosphorylation

Analytical Biochemistry, 2004

Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylatio... more Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite diVerent, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850 cm ¡1 and the attenuation of a peak around 1205 cm ¡1. Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction.

Research paper thumbnail of High-Throughput Flow Cytometry

Drug Discovery, Development, and Manufacturing, 2010

This article describes the creation of a discovery team in an academic environment. The team is s... more This article describes the creation of a discovery team in an academic environment. The team is specifically structured to perform four integrated activities: characterizing membrane protein, formatting the materials for analysis of cell physiology and molecular interactions, performing screens of small-molecule activity on a novel high-throughput flow cytometric instrument platform, and integrating virtual screening with activity screening. These activities improve the overall efficiency of the discovery process. Keywords: discovery team; virtual screening; cell physiology; molecular interactions

Research paper thumbnail of Hight Content-High Throughput drug discovery: Flow cytometry rises to meet the challenge

Research paper thumbnail of Synthesis of o-geranyl (1-thio)diphosphate

Tetrahedron Letters, 1989

ABSTRACT

Research paper thumbnail of Detection of the site of phosphorylation in a peptide using Raman spectroscopy and partial least squares discriminant analysis

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2005

Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13... more Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13-residue peptide whose sequence derives from the cellular protein pp60(c-src) (protein tyrosine kinase). Raman spectra of serine, threonine and tyrosine amino acids and their phosphorylated derivatives are used to aid in the interpretation of peptide spectra. The purity of the synthetic peptides are confirmed by mass spectroscopy. Peptide Raman measurements are performed using the recently reported drop-coating deposition Raman (DCDR) method, followed by Savistky-Golay second derivative (SGSD) pre-processing and multivariate spectral classification using partial least squares (PLS) discriminant analysis. Leave-one-out training/testing results are displayed using a PLS psuedo-probability score plot and shown to facilitate error-free spectral determination of the site of phosphorylation.

Research paper thumbnail of Multiplexed concentration quantification using isotopic surface-enhanced resonance Raman scattering

Journal of Raman Spectroscopy, 2009

The recently developed isotopically edited internal standard approach for surface-enhanced resona... more The recently developed isotopically edited internal standard approach for surface-enhanced resonance Raman scattering (SERRS) based chemical quantification is extended to demonstrate multiplexed detection of four different isotopic variants of a single chromophore. More specifically, it is shown that rhodamine-6G (R6G) with 0, 2, 4, or 6 deuterium substitutions may be reliably quantified in either two-or three-component mixtures. Thus, one isotopic species of known concentration may be used as an internal standard to determine the concentrations of two other isotopic components in a mixture. The concentrations of isotopic R6G SERRS chromophores are determined using partial least squares calibration and shown to yield a predictive accuracy of about ±10% of the total R6G concentration (over 1-50 nM concentration range). These results set the stage for the use of such isotopic variants as tags for the SERRS/SERS quantitation of mixtures containing proteins, peptides, and other compounds.

Research paper thumbnail of Crystallization of human thymidylate synthase

Journal of Molecular Biology, 1991

Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3... more Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A. The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values. The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A.

Research paper thumbnail of Sequence specific cleavage of messenger RNA by a modified ribonuclease H

Bioorganic & Medicinal Chemistry, 1994

... 18. Kawasaki, AM; Casper, MD; Freier, SM; Lesnik, EA; Zounes, MC; Cummins, LL; Gonzalez, C.; ... more ... 18. Kawasaki, AM; Casper, MD; Freier, SM; Lesnik, EA; Zounes, MC; Cummins, LL; Gonzalez, C.; Cook, PDJ Med. ... 21. Rosolen, A.; Kyle, E.; Chavany, C.; Bergan, R.; Kalman, ET; Crouch, R.; Neckers, L. Biochemie 1993, 75, 79-87. ... 25. Carlsson, J.; Drevin, H.; Axen, R. Biochem. ...

Research paper thumbnail of Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase

Archives of Biochemistry and Biophysics, 2014

GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate b... more GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop-and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.

Research paper thumbnail of Targeting PCNA Phosphorylation in Breast Cancer

Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.

Research paper thumbnail of NMR structure of a DNA duplex containing nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the unmodified control

Nucleic Acids Research, 2000

Research paper thumbnail of 2.444 Suppression of neurotoxicity by antioxidant molecules in cellular models of Parkinson's disease

Parkinsonism & Related Disorders, 2007

Research paper thumbnail of Exploratory studies on azole carboxamides as nucleobase analogs: thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole-3-carboxamide

Nucleic Acids Research, 1998

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog... more In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2′-deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2′-deoxy-3′,5′-di-O-p-toluoyl-β-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O-toluoyl-D-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2′-Deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T > dG > dA > dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syn τ versus anti τ conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized anti τ and syn τ conformations are twisted out of the plane of the pyrrole ring by 6-14_. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).

Research paper thumbnail of Quantitative LC–MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor

Analytical Biochemistry, 2013

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing ... more An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/ll for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), c-aminobutyric acid (NAGABA), and glycine (NAGly);

Research paper thumbnail of Computational analysis of high-throughput flow cytometry data

Expert Opinion on Drug Discovery, 2012

Introduction: Flow cytometry has been around for over 40 years, but only recently has the opportu... more Introduction: Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered: Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion: There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible.

Research paper thumbnail of NMR structure analysis of the DNA nine base pair duplex D(CATGAGTAC) D(GTAC(NP3)CATG)

Research paper thumbnail of Prediction of drug toxicity in HepG2 cells using laser scanning cytometry

Toxicology Letters, 2010

(24) and 335-192 (47)]; CLZ [(m/z 327-270 (25) and 327-192 (44)]; NCLZ [(m/z 313-270 (25) and 313... more (24) and 335-192 (47)]; CLZ [(m/z 327-270 (25) and 327-192 (44)]; NCLZ [(m/z 313-270 (25) and 313-192 (41)]. Mobile phase constituted of 50% methanol with 0.1% formic acid. The flow rate is 0.6 mL/min. Total analysis time is 1.5 min. A group of 200 clinical plasma samples were analyzed and results compared with LC-MS/MS methods. Limit of detection was 1 ng/mL for CLZ and NCLZ. Limit of quantification was 5 ng/mL. The within run precision and accuracy for QC samples at 150, 300 and 600 ng/mL were between 3.69-10.11% and −0.01% to −10.31%, respectively. The correlation (r 2) between the FIA-MS/MS procedure and LC-MS/MS procedure was greater than 0.98. The FIA-MS/MS procedure was capable of analyzing CLZ and NCLZ in human plasma samples efficiently and accurately.

Research paper thumbnail of Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Nucleosides, Nucleotides & Nucleic Acids, 2005

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2&am... more Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

Research paper thumbnail of Mechanism-based inactivation of Escherichia coli .beta.-hydroxydecanoyl thiol ester dehydrase: assignment of the imidazole nitrogen-15 NMR resonances and determination of the structure of the alkylated histidine

Journal of the American Chemical Society, 1993

Nitrogen-1 5 nuclear magnetic resonance spectroscopy was used to determine the structure of the a... more Nitrogen-1 5 nuclear magnetic resonance spectroscopy was used to determine the structure of the active-site histidine-70 adduct formed when @-hydroxydecanoyl thiol ester dehydrase from Escherichia coli reacts with the mechanism-based inactivator S-(3-decynoyl)-N-acetylcysteamine (3-decynoyl-NAC). In order to obtain the amount of labeled enzyme necessary for spectral studies, thefabA gene, which encodes dehydrase, was overexpressed to give dehydrase as 1520% of soluble protein. To simplify the interpretation of the NMR spectra, the non-active-site histidine residue His-129 was converted to an asparagine residue using site-directed mutagenesis. The specific activity and response of the mutant to 3-decynoyl-NAC are unaltered. It is known that type @ ('pyridine-like") nitrogens in imidazoles resonate 60-80 ppm downfield of type a ('pyrrole-like") nitrogens. To assign the imidazole nitrogen resonances in dehydrase, wild-type and mutant enzymes were labeled with I5NH4Cl or with ['5N6']histidine. Analysis of the I5N NMR spectra allowed the assignment of the resonances of the imidazole nitrogens of His-129 and His-70. For His-70, the spectra show that N*' resonates upfield of Nf2 in the native enzyme and is therefore a type a nitrogen. In the inactivated enzyme the signals are reversed, and Nf2 is a type a nitrogen. These results demonstrate that Nf2 of His-70 becomes alkylated upon inactivation of dehydrase with 3-decynoyl-NAC and thus is the probable locus of active-site basicity in the normal reactions catalyzed by dehydrase. In addition, the imidazole nitrogen chemical shifts suggest that N*' may be involved in a hydrogen bond in native dehydrase but that Nf2 is not. The mechanistic implications of this are discussed. (I) Bloch, K. In The Enzymes, 3rd 4.

Research paper thumbnail of Stereochemical course of the Escherichia coli imidazole glycerol phosphate dehydratase reaction

Journal of the American Chemical Society, 1993

The va,st majority of enzyme-catalyzed dehydrations involve suhstretes wit,h the departing hydrog... more The va,st majority of enzyme-catalyzed dehydrations involve suhstretes wit,h the departing hydrogen and hydroxyl group sit.iiated a and 6, respectively, to a carbonyl or an imine group.' (2 5) Hansen, P. E. In Annual Reports on N M R Spectroscopy; Webb, G.

Research paper thumbnail of The Raman detection of peptide tyrosine phosphorylation

Analytical Biochemistry, 2004

Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylatio... more Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite diVerent, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850 cm ¡1 and the attenuation of a peak around 1205 cm ¡1. Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction.

Research paper thumbnail of High-Throughput Flow Cytometry

Drug Discovery, Development, and Manufacturing, 2010

This article describes the creation of a discovery team in an academic environment. The team is s... more This article describes the creation of a discovery team in an academic environment. The team is specifically structured to perform four integrated activities: characterizing membrane protein, formatting the materials for analysis of cell physiology and molecular interactions, performing screens of small-molecule activity on a novel high-throughput flow cytometric instrument platform, and integrating virtual screening with activity screening. These activities improve the overall efficiency of the discovery process. Keywords: discovery team; virtual screening; cell physiology; molecular interactions

Research paper thumbnail of Hight Content-High Throughput drug discovery: Flow cytometry rises to meet the challenge

Research paper thumbnail of Synthesis of o-geranyl (1-thio)diphosphate

Tetrahedron Letters, 1989

ABSTRACT

Research paper thumbnail of Detection of the site of phosphorylation in a peptide using Raman spectroscopy and partial least squares discriminant analysis

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2005

Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13... more Normal (non-enhanced) Raman spectroscopy is used to determine the site of phosphorylation on a 13-residue peptide whose sequence derives from the cellular protein pp60(c-src) (protein tyrosine kinase). Raman spectra of serine, threonine and tyrosine amino acids and their phosphorylated derivatives are used to aid in the interpretation of peptide spectra. The purity of the synthetic peptides are confirmed by mass spectroscopy. Peptide Raman measurements are performed using the recently reported drop-coating deposition Raman (DCDR) method, followed by Savistky-Golay second derivative (SGSD) pre-processing and multivariate spectral classification using partial least squares (PLS) discriminant analysis. Leave-one-out training/testing results are displayed using a PLS psuedo-probability score plot and shown to facilitate error-free spectral determination of the site of phosphorylation.

Research paper thumbnail of Multiplexed concentration quantification using isotopic surface-enhanced resonance Raman scattering

Journal of Raman Spectroscopy, 2009

The recently developed isotopically edited internal standard approach for surface-enhanced resona... more The recently developed isotopically edited internal standard approach for surface-enhanced resonance Raman scattering (SERRS) based chemical quantification is extended to demonstrate multiplexed detection of four different isotopic variants of a single chromophore. More specifically, it is shown that rhodamine-6G (R6G) with 0, 2, 4, or 6 deuterium substitutions may be reliably quantified in either two-or three-component mixtures. Thus, one isotopic species of known concentration may be used as an internal standard to determine the concentrations of two other isotopic components in a mixture. The concentrations of isotopic R6G SERRS chromophores are determined using partial least squares calibration and shown to yield a predictive accuracy of about ±10% of the total R6G concentration (over 1-50 nM concentration range). These results set the stage for the use of such isotopic variants as tags for the SERRS/SERS quantitation of mixtures containing proteins, peptides, and other compounds.

Research paper thumbnail of Crystallization of human thymidylate synthase

Journal of Molecular Biology, 1991

Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3... more Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A. The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values. The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A.

Research paper thumbnail of Sequence specific cleavage of messenger RNA by a modified ribonuclease H

Bioorganic & Medicinal Chemistry, 1994

... 18. Kawasaki, AM; Casper, MD; Freier, SM; Lesnik, EA; Zounes, MC; Cummins, LL; Gonzalez, C.; ... more ... 18. Kawasaki, AM; Casper, MD; Freier, SM; Lesnik, EA; Zounes, MC; Cummins, LL; Gonzalez, C.; Cook, PDJ Med. ... 21. Rosolen, A.; Kyle, E.; Chavany, C.; Bergan, R.; Kalman, ET; Crouch, R.; Neckers, L. Biochemie 1993, 75, 79-87. ... 25. Carlsson, J.; Drevin, H.; Axen, R. Biochem. ...

Research paper thumbnail of Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase

Archives of Biochemistry and Biophysics, 2014

GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate b... more GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop-and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.

Research paper thumbnail of Targeting PCNA Phosphorylation in Breast Cancer

Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.

Research paper thumbnail of NMR structure of a DNA duplex containing nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the unmodified control

Nucleic Acids Research, 2000

Research paper thumbnail of 2.444 Suppression of neurotoxicity by antioxidant molecules in cellular models of Parkinson's disease

Parkinsonism & Related Disorders, 2007

Research paper thumbnail of Exploratory studies on azole carboxamides as nucleobase analogs: thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole-3-carboxamide

Nucleic Acids Research, 1998

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog... more In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2′-deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2′-deoxy-3′,5′-di-O-p-toluoyl-β-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O-toluoyl-D-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2′-Deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T > dG > dA > dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syn τ versus anti τ conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized anti τ and syn τ conformations are twisted out of the plane of the pyrrole ring by 6-14_. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).

Research paper thumbnail of Quantitative LC–MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor

Analytical Biochemistry, 2013

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing ... more An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/ll for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), c-aminobutyric acid (NAGABA), and glycine (NAGly);

Research paper thumbnail of Computational analysis of high-throughput flow cytometry data

Expert Opinion on Drug Discovery, 2012

Introduction: Flow cytometry has been around for over 40 years, but only recently has the opportu... more Introduction: Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered: Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion: There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible.