David Stranz - Academia.edu (original) (raw)

Papers by David Stranz

cAMP-dependent protein kinase (cAPK) is a key component in numerous cell signaling pathways. The ... more cAMP-dependent protein kinase (cAPK) is a key component in numerous cell signaling pathways. The cAPK regulatory (R) subunit maintains the kinase in an inactive state until cAMP saturation of the R-subunit leads to activation of the enzyme. To delineate the conformational changes associated with cAPK activation, the amide hydrogen/deuterium exchange in the cAPK type IIβ R-subunit was probed by electrospray

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory... more Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R 2 C 2 ). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/docking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMPfree state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R -C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMPdomains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIa and the previously studied RIIb upon cAMP-binding suggests isoform-specific differences in cAMPdependent regulation of PKA activity.

Journal of biomolecular techniques : JBT, 2003

An automated approach for the rapid analysis of protein structure has been developed and used to ... more An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognize...

Spectrochimica Acta Part A: Molecular Spectroscopy, 1980

Toxicology Mechanisms and Methods, 2008

ABSTRACT As high-throughput technologies have developed in the pharmaceutical industry, the deman... more ABSTRACT As high-throughput technologies have developed in the pharmaceutical industry, the demand for identification of possible metabolites using predominantly liquid chromatographic/mass spectrometry-mass spectrometry/mass spectrometry (LC/MS-MS/MS) for a large number of molecules in drug discovery has also increased. In parallel, computational technologies have also been developed to generate predictions for metabolites alongside methods to predict MS spectra and score the quality of the match with experimental spectra. The goal of the current study was to generate metabolite predictions from molecular structure with a software product, MetaDrug. In vitro microsomal incubations were used to ultimately produce MS data that could be used to verify the predictions with Apex, which is a new software tool that can predict the molecular ion spectrum and a fragmentation spectrum, automating the detailed examination of both MS and MS/MS spectra. For the test molecule imipramine used to illustrate the combined in vitro/in silico process proposed, MetaDrug predicts 16 metabolites. Following rat microsomal incubations with imipramine and analysis of the MS(n) data using the Apex software, strong evidence was found for imipramine and five metabolites and weaker evidence for five additional metabolites. This study suggests a new approach to streamline MS data analysis using a combination of predictive computational approaches with software capable of comparing the predicted metabolite output with empirical data when looking at drug metabolites.

Protein Science, 2005

hydrogen/deuterium exchange subunit isoforms of protein kinase A revealed by amide Distinct inter... more hydrogen/deuterium exchange subunit isoforms of protein kinase A revealed by amide Distinct interaction modes of an AKAP bound to two regulatory data Supplementary http://www.proteinscience.org/cgi/content/full/ps.051687305/DC1 "Supplemental Research Data" Abstract

Protein Science, 2003

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dime... more cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIbeta subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.

Proceedings of the National Academy of Sciences, 2003

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used t... more An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

Proceedings of the National Academy of Sciences, 2009

Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) ... more Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) in the circulation and governs their biogenesis, metabolism, and functional interactions. To decipher these important structure-function relationships, it will be necessary to understand the structure, stability, and plasticity of the apoA-I molecule. Biophysical studies show that lipid-free apoA-I contains a large amount of ␣-helical structure but the location of this structure and its properties are not established. We used hydrogen-deuterium exchange coupled with a fragmentation-separation method and mass spectrometric analysis to study human lipid-free apoA-I in its physiologically pertinent monomeric form. The acquisition of Ϸ100 overlapping peptide fragments that redundantly cover the 243-residue apoA-I polypeptide made it possible to define the positions and stabilities of helical segments and to draw inferences about their interactions and dynamic properties. Residues 7-44, 54 -65, 70 -78, 81-115, and 147-178 form ␣-helices, accounting for a helical content of 48 ؎ 3%, in agreement with circular dichroism measurements (49%). At 3 to 5 kcal/mol in free energy of stabilization, the helices are far more stable than could be achieved in isolation, indicating mutually stabilizing helix bundle interactions. However the helical structure is dynamic, unfolding and refolding in seconds, allowing facile apoA-I reorganization during HDL particle formation and remodeling.

Journal of Molecular Biology, 2003

Journal of Molecular Biology, 2002

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate fun... more Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchangemass spectrometry (DXMS) to probe conformational changes on Csk in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire Csk polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that Csk adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the g phosphate group of ATP induces unique local and long-range conformational changes in Csk that may influence regulatory motions in the catalytic pathway.

Journal of Molecular Biology, 2004

The Journal of Chemical Physics, 1981

Matrix isolation techniques are used to investigate the spectra of lead molecules and, in particu... more Matrix isolation techniques are used to investigate the spectra of lead molecules and, in particular, to obtain resonance Raman spectra of lead vapors isolated in solid xenon matrices. The presence of Pb2 is confirmed by the visible adsorption, and Raman spectra yield a vibrational frequency for the ground state of 108 per cm and a dissociation energy of 8200 per cm. A second resonance Raman progression indicates a Pb3 species of D3h symmetry. Finally, two additional Raman features at approximately 111 per cm spacing are evidence for a third species, tentatively identified as Pb4.

Pesticide Science, 1990

which the chlorine atom is substituted at diSferent positions of the pyridine ring have widely va... more which the chlorine atom is substituted at diSferent positions of the pyridine ring have widely varying biological properties. The 3-chloro analog (I) is a post-emergence and pre-emergence herbicide, the 4-and 5-chloro analogs (11, III) ure post-emergence herbicides but not pre-emergence, and the 6-chloro analog ( I V ) is inactive. Computer graphic and molecular mechanics analyses of their molecular conformations showed that the 4-and 5-chloro analogs adopt a coplanar, intramoleculurly hydrogen bonded conformation whereas the 3-chloro unalog does not. High-level quantum mechanical calculations of the conformational preferences of related model systems were in agreement with these results. Based on this, 11 and III were predicted to have higher octunollwater partition coeficients relative to I, leading to higher soil binding and weaker xylem transport, hence their observed weaker pre-emergence activities. Experimental measurements of octanollwater purtition coeflciznts, soil binding, and infrared hydrogen bonding studies verified these predictions. Molecular modeling techniques are usually used for designing compounds to fit enzyme active sites and designing putative receptor models. This study demonstrates the usefulness of these techniques for dealing with transport problems. 49 Pesric. Sci. 0031-613X/90/$03.50 0 1990 Society of Chemical Industry. Printed in Great Britain T. A. A~7drea et al. 50