Pulsed dye laser-induced europium (III) and terbium (III) ion luminescence probe experiments for the study of the structure and function of calcium-binding proteins will be further developed and exploited. With the capability of measuring metal ion binding in the namomolar concentration range a general, site-specific method of determining the thermodynamic parameters of metal ion-protein interactions will be established. Calmodulin, the ubiquitous intracellular calcium-modulated regulatory protein, will be extensively studied with regard to its metal ion binding properties and its interaction with target enzymes and drug molecules. These studies will be aided by a newly developed, continuous, spectrophotometric assay for calmodulin based on the activation of NAD kinase. The calmodulin-NAD kinase interaction will be probed in depth. Laser spectroscopic experiments will be carried out on other calcium-modulated proteins including parvalbumin and calcineurin as well as on phospholipase A2, glutamine synthetase, and proteins of the blood coagulation cascade through collaborative arrangements. Luminescence or fluorescence-monitored stopped-flow kinetic studies of metal ion disociation from calmodulin and other calcium-modulated proteins will be carried out. The role of metal ion binding in the dynamics of protein folding or unfolding will be examined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023599-14
Application #
3271771
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1977-01-01
Project End
1990-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
14
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Supkowski, Ronald M; DeW Horrocks Jr, William (2003) Lanthanide ions as probes of electron transfer in proteins. Met Ions Biol Syst 40:281-322
Chaudhuri, D; Horrocks Jr, W D; Amburgey, J C et al. (1997) Characterization of lanthanide ion binding to the EF-hand protein S100 beta by luminescence spectroscopy. Biochemistry 36:9674-80
Bruno, J; Horrocks Jr, W D; Beckingham, K (1996) Characterization of Eu(III) binding to a series of calmodulin binding site mutants using laser-induced Eu(III) luminescence spectroscopy. Biophys Chem 63:1-16
Reynaldo, L P; Villafranca, J J; Horrocks Jr, W D (1996) Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy. Protein Sci 5:2532-44
Frey, M W; Frey, S T; Horrocks Jr, W D et al. (1996) Elucidation of the metal-binding properties of the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase by lanthanide(III) luminescence spectroscopy. Chem Biol 3:393-403
Burroughs, S E; Horrocks Jr, W D; Ren, H et al. (1994) Characterization of the lanthanide ion-binding properties of calcineurin-B using laser-induced luminescence spectroscopy. Biochemistry 33:10428-36
Horrocks Jr, W D (1993) Luminescence spectroscopy. Methods Enzymol 226:495-538
Cronce, D T; Horrocks Jr, W D (1992) Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer. Biochemistry 31:7963-9
Amann, B T; Mulqueen, P; Horrocks Jr, W D (1992) A continuous spectrophotometric assay for the activation of plant NAD kinase by calmodulin, calcium(II), and europium(III) ions. J Biochem Biophys Methods 25:207-17
Bruno, J; Horrocks Jr, W D; Zauhar, R J (1992) Europium(III) luminescence and tyrosine to terbium(III) energy-transfer studies of invertebrate (octopus) calmodulin. Biochemistry 31:7016-26

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