This project applies techniques for fast time-resolved magnetic circular dichroism (TRMCD), natural circular dichroism (TRCD, and ordinary absorption spectroscopies to the study of function in heme proteins and folding in heme proteins and small peptides. The novel optical methods employed use near-null ellipsometry and polarimetry to study rapid kinetic processes (nanosecond to seconds) in biomolecules that contain magneto-optically active chromophores, such as heme and the aromatic amino acids, and naturally chiral chromophores, such as the amide groups of proteins and peptides. These techniques will be used to identify and study the earliest (submillisecond) events in the folding reactions of heme proteins such as cytochrome c. A major goal is the determination of a parameter that is fundamental to understanding the nature of protein folding: the speed with which the different unfolded conformations interconvert with one another. If this is slow compared to folding itself, then understanding protein folding will require more complicated theories (e.g., energy landscape) than the transition state theory used for typical chemical reactions. Such understanding may ultimately prove helpful in developing therapies for the many diseases associated with protein misfolding, such as cystic fibrosis, type 2 diabetes, and Alzheimer's, Parkinson's, and Creutzfeldt- Jakob disease. A major goal of the functional studies is to understand how the four subunits that make up the hemoglobin molecule cooperate with each other to transport oxygen more efficiently. A recent hypothesis about this cooperativity (Ackers symmetry rule), based originally on thermodynamic measurements, is tested by kinetic measurements in this project. A novel model for hemoglobin allostery, emerging from this linkage of thermodynamics and kinetics, holds promise for simplifying and systematizing our understanding of hemoglobin's dynamics and its control in the body by allosteric effectors such as organic phosphates. In addition, TRMCD studies of the aromatic amino acid residue tryptophan [337, positioned at a site critical for cooperativity, are intended to further clarify how hemoglobin's subunits work together as an efficient """"""""molecular machine"""""""" for transporting oxygen from the lungs to the tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
2R01EB002056-17A1
Application #
6874802
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Zhang, Yantian
Project Start
2004-09-01
Project End
2008-07-31
Budget Start
2004-09-01
Budget End
2005-07-31
Support Year
17
Fiscal Year
2004
Total Cost
$325,897
Indirect Cost
Name
University of California Santa Cruz
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Kliger, David S; Chen, Eefei; Goldbeck, Robert A (2012) Probing kinetic mechanisms of protein function and folding with time-resolved natural and magnetic chiroptical spectroscopies. Int J Mol Sci 13:683-97
Chen, Eefei; Goldbeck, Robert A; Kliger, David S (2010) Nanosecond time-resolved polarization spectroscopies: tools for probing protein reaction mechanisms. Methods 52:3-11
Esquerra, Raymond M; López-Peña, Ignacio; Tipgunlakant, Pooncharas et al. (2010) Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics. Phys Chem Chem Phys 12:10270-8
Goldbeck, Robert A; Chen, Eefei; Kliger, David S (2009) Early events, kinetic intermediates and the mechanism of protein folding in cytochrome C. Int J Mol Sci 10:1476-99
Goldbeck, Robert A; Pillsbury, Marlisa L; Jensen, Russell A et al. (2009) Optical detection of disordered water within a protein cavity. J Am Chem Soc 131:12265-72
Chen, Eefei; Goldbeck, Robert A; Kliger, David S (2009) Probing early events in ferrous cytochrome c folding with time-resolved natural and magnetic circular dichroism spectroscopies. Curr Protein Pept Sci 10:464-75
Esquerra, Raymond M; Jensen, Russell A; Bhaskaran, Shyam et al. (2008) The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin. J Biol Chem 283:14165-75
Chen, Eefei; Van Vranken, Vanessa; Kliger, David S (2008) The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c. Biochemistry 47:5450-9
Williams, Terrie M; Zavanelli, Mary; Miller, Melissa A et al. (2008) Running, swimming and diving modifies neuroprotecting globins in the mammalian brain. Proc Biol Sci 275:751-8
Thompson, Alisha M; Reddi, Amit R; Shi, Xiaoli et al. (2007) Measurement of the heme affinity for yeast dap1p, and its importance in cellular function. Biochemistry 46:14629-37

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