Faculty Collaboration Database

Multiquantum EPR spectroscopy of spin-labeled arrestin K267C at 35 GHz. Biophys J 2005 May;88(5):3641-7

Date

03/08/2005

Pubmed ID

15749769

Pubmed Central ID

PMC1305511

DOI

10.1529/biophysj.104.054924

Scopus ID

2-s2.0-17844402249 (requires institutional sign-in at Scopus site)   19 Citations

Abstract

Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics.

Author List

Klug CS, Camenisch TG, Hubbell WL, Hyde JS

Author

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Absorption
Animals
Arrestins
Biophysics
Cattle
Electron Spin Resonance Spectroscopy
Escherichia coli
Microwaves
Models, Molecular
Proteins
Quantum Theory
Retinal Rod Photoreceptor Cells
Sensitivity and Specificity
Signal Processing, Computer-Assisted
Spin Labels
Spin Trapping
Temperature
Time Factors