This grant will support a continuing research program on protein dynamics, using ultraviolet resonance Raman (UVRR) spectroscopy to characterize protein motions in real time. Understanding proteins as dynamic machines as opposed to rigid structures is key to many potential applications in medicinal chemistry and biotechnology. The site- specific and spatially resolved spectroscopic technique of UVRR will be brought to bear on a wide range of protein mechanisms, from subtly complex folding pathways to functional dynamics such as pore closure and allostery. Folding motions will be investigated in small proteins with different types of secondary structures: CspA (all-? sheet), Villin Headpiece Domain 36 (all-? helix) and FSD (a designed miniprotein with a ?-?-? fold). Functional dynamics will be probed using key models of biological relevance;ferritin (pore/loop closure), ADH (catalysis-related motions), calmodulin (ligand-binding motions) and PTP-ase (loop dynamics). These diverse motions, involving a range of substructures, will support a search for common dynamical principles. Static and time-resolved UVRR experiments will be supported by simultaneous fluorescence monitoring, as well as computational molecular dynamics simulations and static nuclear magnetic resonance, circular dichroism, and UV-Vis spectroscopy.

Public Health Relevance

Proteins are dynamic macromolecules which must be understood at a dynamic as well as structural level in order to tackle biologically relevant problems. Types of protein motions include (un)folding, loop, hinge and pore motions and allostery. All are relevant to a wide array of diseases and disorders caused by protein malfunction. The mechanisms of these motions are amenable to characterization by UV Resonance Raman spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025158-36
Application #
8535158
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Smith, Ward
Project Start
1978-06-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
36
Fiscal Year
2013
Total Cost
$341,138
Indirect Cost
$120,337
Name
University of Washington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Jones, Eric M; Monza, Emanuele; Balakrishnan, Gurusamy et al. (2014) Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study. J Am Chem Soc 136:10325-39
Balakrishnan, Gurusamy; Soldatova, Alexandra V; Reid, Philip J et al. (2014) Ultrafast charge transfer in nickel phthalocyanine probed by femtosecond Raman-induced Kerr effect spectroscopy. J Am Chem Soc 136:8746-54
Jones, Eric M; Balakrishnan, Gurusamy; Squier, Thomas C et al. (2014) Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy. Protein Sci 23:1094-101
Soldatova, Alexandra V; Ibrahim, Mohammed; Spiro, Thomas G (2013) Electronic structure and ligand vibrations in FeNO, CoNO, and FeOO porphyrin adducts. Inorg Chem 52:7478-86
Spiro, Thomas G; Soldatova, Alexandra V; Balakrishnan, Gurusamy (2013) CO, NO and O2 as Vibrational Probes of Heme Protein Interactions. Coord Chem Rev 257:511-527
Balakrishnan, Gurusamy; Jarzecki, Andrzej A; Wu, Qiang et al. (2012) Mode recognition in UV resonance Raman spectra of imidazole: histidine monitoring in proteins. J Phys Chem B 116:9387-95
Balakrishnan, Gurusamy; Hu, Ying; Spiro, Thomas G (2012) His26 protonation in cytochrome c triggers microsecond β-sheet formation and heme exposure: implications for apoptosis. J Am Chem Soc 134:19061-9
Jones, Eric M; Balakrishnan, Gurusamy; Spiro, Thomas G (2012) Heme reactivity is uncoupled from quaternary structure in gel-encapsulated hemoglobin: a resonance Raman spectroscopic study. J Am Chem Soc 134:3461-71
Spiro, Thomas G; Soldatova, Alexandra V (2012) Ambidentate H-bonding of NO and O2 in heme proteins. J Inorg Biochem 115:204-10
Soldatova, Alexandra V; Butterfield, Cristina; Oyerinde, Oyeyemi F et al. (2012) Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO(2). J Biol Inorg Chem 17:1151-8

Showing the most recent 10 out of 59 publications