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

• Published in: Protein science Vol. 9; no. 2; pp. 302 - 309
• Main Authors: GAPONENKO, VADIM, HOWARTH, JACK W., COLUMBUS, LINDA, GASMI-SEABROOK, GENEVIEVE, YUAN, JIE, HUBBELL, WAYNE L., ROSEVEAR, PAUL R.
• Format: Journal Article
• Language: English
• Published: Bristol Cambridge University Press 01.02.2000; Cold Spring Harbor Laboratory Press
• Subjects: Amino Acid Substitution; barnase; Cysteine - chemistry; Electron Spin Resonance Spectroscopy; EPR; Magnetic Resonance Spectroscopy; Models, Molecular; Mutagenesis, Site-Directed; Nitrogen Isotopes; NMR; paramagnetic enhancement; Protein Folding; protein global fold; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Ribonucleases - chemistry; Ribonucleases - genetics; site‐directed spin labeling; Spin Labels; Thermodynamics; NMR; barnase; paramagnetic enhancement; protein global fold; site-directed spin labeling; EPR
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.9.2.302

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.