Time-resolved infrared (TRIR) studies of protein dynamics on all timescales from subpicosecond to seconds are proposed. The primary targets of this study are cytochrome oxidases from mammals and bacteria. Some work on myoglobin and hemoglobin is suggested to establish technical feasibility or to model the more complicated oxidases. The long-term goal is to understand in structural detail the functional dynamics of the oxidases, upon which all aerobic life on earth depends. A technical objective is to develop new and generally applicable approaches to the study of protein dynamics, based upon the observation by infrared of structural features that are invisible to other spectroscopic probes. A suite of TRIR techniques will be used: optical delay methods for subpicosecond to nanosecond dynamics; real-time TRIR for dynamics from nanoseconds to seconds; time-resolved infrared linear dichroism for direct determination of the relative orientation of infrared-active structures and electronic dipoles, and step-scan time-resolved FAIR for observation of transient phenomena over the entire infrared spectrum. These studies will be supplemented where appropriate by transient electronic spectroscopy, time- resolved magnetic circular dichroism, and time-resolved resonance Raman investigations. Specific studies to be performed include: (1) TRIR investigation of ligation dynamics, including photodissociation/recombination of heme-bond ligands, fate of ligands after photodissociation, binding to nonheme metals, and dependence of dynamics upon steric and electronic factors. The result will be detailed understanding of ligation and steric phenomena at the site of O2 activation. (2) TRIR studies of endogenous protein IR chromophores, including amino acid side chains, the peptide backbone and prosthetic groups. The results will elucidate the response of the protein structure and conformation to ligation and redox perturbations. (3) Time-resolved infrared linear dichroism, studied in connection with (1) and (2) above, aimed at determining specific structural reorientations which occur during the short-timescale dynamics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045807-03
Application #
3305252
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1991-05-01
Project End
1995-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Los Alamos National Lab
Department
Type
Organized Research Units
DUNS #
City
Los Alamos
State
NM
Country
United States
Zip Code
87545
Bailey, James A; Tomson, Farol L; Mecklenburg, Sandra L et al. (2002) Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo(3) from Escherichia coli. Biochemistry 41:2675-83
Callender, R H; Dyer, R B; Gilmanshin, R et al. (1998) Fast events in protein folding: the time evolution of primary processes. Annu Rev Phys Chem 49:173-202
Gilmanshin, R; Williams, S; Callender, R H et al. (1997) Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc Natl Acad Sci U S A 94:3709-13
Puustinen, A; Bailey, J A; Dyer, R B et al. (1997) Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli. Biochemistry 36:13195-200
Dyer, R B; Peterson, K A; Stoutland, P O et al. (1994) Picosecond infrared study of the photodynamics of carbonmonoxy-cytochrome c oxidase. Biochemistry 33:500-7
Causgrove, T P; Dyer, R B (1993) Protein response to photodissociation of CO from carbonmonoxymyoglobin probed by time-resolved infrared spectroscopy of the amide I band. Biochemistry 32:11985-91