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

We describe a simple experimental approach for the rapid determination of p rotein global folds. This strategy utilizes site-directed spin labeling (SD SL) 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 e ffects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were N-15 e nriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide H-1 longitudinal relaxatio n 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 proce sses at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Angstrom w ere calculated from the magnitude of the paramagnetic contribution to the r elaxation processes and individual amide H-1 correlation times. Distance re straints from the nitroxide spin to amide protons were used as restraints i n structure calculations. Using nitroxide to amide H-1 distances as long-ra nge restraints and known secondary structure restraints, barnase global fol ds were calculated having backbone RMSDs <3 Angstrom from the crystal struc ture. This approach makes it possible to rapidly obtain the overall topolog y of a protein using a limited number of paramagnetic distance restraints.