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Protein global fold determination using site-directed spin and isotope labeling

Published online by Cambridge University Press:  01 February 2000

VADIM GAPONENKO ,
JACK W. HOWARTH ,
LINDA COLUMBUS ,
GENEVIEVE GASMI-SEABROOK ,
JIE YUAN ,
WAYNE L. HUBBELL  and
PAUL R. ROSEVEAR
VADIM GAPONENKO
Affiliation:
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
JACK W. HOWARTH
Affiliation:
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
LINDA COLUMBUS
Affiliation:
Departments of Chemistry and Biochemistry and the Jules Stein Eye Institute, University of California, Los Angeles, California 90095
GENEVIEVE GASMI-SEABROOK
Affiliation:
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
JIE YUAN
Affiliation:
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
WAYNE L. HUBBELL
Affiliation:
Departments of Chemistry and Biochemistry and the Jules Stein Eye Institute, University of California, Los Angeles, California 90095
PAUL R. ROSEVEAR
Affiliation:
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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Abstract

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Å were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 Å from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Keywords

barnaseEPRNMRparamagnetic enhancementprotein global foldsite-directed spin labeling
Type
Research Article
Information
Protein Science , Volume 9 , Issue 2 , February 2000 , pp. 302 - 309
Copyright
© 2000 The Protein Society

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