Abstract

In this project proposal, the folding and unfolding reactions of the model system recombinant alpha-macrophage colony stimulating factor beta (rhm-CSFbeta) will be characterized. Stopped flow CD will be used to analyze the kinetics of the formation of the first detectable kinetic intermediate on the refolding pathway (monomeric rhm-CSFbeta with reformed secondary structure) and the effects of carboxamido-methylation of thiols on the regaining of secondary structure. HPLC and fluorescence intensity changes will be used to examine the refolding process in the slow time domain (minutes-hours) where the effect of parameters such as protein concentration on the refolding pathway will be examined. The time- dependence of deuterium/hydrogen exchanges kinetics for discrete rhm- CSFbeta domains during unfolding will be determined by electrospray mass spectrometry on peptic peptides generated after rapid-mix-quench exchange experiments. These experiments should permit mapping of the kinetic pathway of rhm-MCSFbeta folding and a detailed analysis of the first detectable kinetic step in the pathway as well as a correlation between solvent accessible protein domains and the formation/decomposition of kinetic intermediates. The effects of chemical insult due to alkylation and oxidation of thiols and oxidation of methionine on the energetics on folding kinetics will be analyzed and correlated with the site of modifications.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Program Projects (P01)
Project #
5P01ES000040-38
Application #
6564404
Study Section
Project Start
2001-12-01
Project End
2002-11-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
38
Fiscal Year
2002
Total Cost
$178,522
Indirect Cost

  Institution

Name
Oregon State University
Department
Type
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339

  Publications

Ehrlich, Allison K; Pennington, Jamie M; Tilton, Susan et al. (2017) AhR activation increases IL-2 production by alloreactive CD4+ T cells initiating the differentiation of mucosal-homing Tim3+ Lag3+ Tr1 cells. Eur J Immunol 47:1989-2001
Ehrlich, Allison K; Pennington, Jamie M; Wang, Xisheng et al. (2016) Activation of the Aryl Hydrocarbon Receptor by 10-Cl-BBQ Prevents Insulitis and Effector T Cell Development Independently of Foxp3+ Regulatory T Cells in Nonobese Diabetic Mice. J Immunol 196:264-73
Venkataraman, Anand; Coleman, Daniel J; Nevrivy, Daniel J et al. (2014) Grp1-associated scaffold protein regulates skin homeostasis after ultraviolet irradiation. Photochem Photobiol Sci 13:531-40
Viera, Liliana; Radmilovich, Milka; Vargas, Marcelo R et al. (2013) Temporal patterns of tyrosine nitration in embryo heart development. Free Radic Biol Med 55:101-8
Venkataraman, Anand; Nevrivy, Daniel J; Filtz, Theresa M et al. (2012) Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway. Cell Biol Int 36:1115-28
Traber, Maret G; Mustacich, Debbie J; Sullivan, Laura C et al. (2010) Vitamin E status and metabolism in adult and aged aryl hydrocarbon receptor null mice. J Nutr Biochem 21:1193-9
Marshall, Nikki B; Kerkvliet, Nancy I (2010) Dioxin and immune regulation: emerging role of aryl hydrocarbon receptor in the generation of regulatory T cells. Ann N Y Acad Sci 1183:25-37
Vassallo, Jeffrey D; Kaetzel, Rhonda S; Born, Stephanie L et al. (2010) Gamma-glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity. Food Chem Toxicol 48:1612-8
Beckman, Joseph S (2009) Understanding peroxynitrite biochemistry and its potential for treating human diseases. Arch Biochem Biophys 484:114-6
Harder, Mark E; Malencik, Dean A; Yan, Xuguang et al. (2009) Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate. Biophys Chem 141:1-10

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