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A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data
Nicolas L Fawzi1, Mark R Fleissner, Nicholas J Anthis
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA 20892-0520, USA.
A new spin label, R1p, reduces internal motion for more accurate paramagnetic relaxation enhancement (PRE) studies in biomolecules. This simplifies distance measurements and analysis of protein dynamics.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Paramagnetic relaxation enhancement (PRE) measures distances in biomolecules.
- Flexible spin labels complicate PRE data interpretation due to paramagnetic center delocalization.
Purpose of the Study:
- Introduce a novel, disulfide-linked nitroxide spin label, R1p.
- Overcome limitations of flexible spin labels in PRE studies.
- Simplify quantitative interpretation of PRE data.
Main Methods:
- Developed and utilized a novel R1p spin label.
- Performed Electron Paramagnetic Resonance (EPR) measurements.
- Studied calmodulin (CaM) and T4 lysozyme (T4L) proteins.
Main Results:
- R1p exhibited significantly reduced internal motion compared to the R1 spin label (MTSL).
- A single nitroxide position explained PRE data for R1p-labeled proteins.
- Multiple conformations were needed for R1-labeled proteins.
Conclusions:
- R1p provides a fixed nitroxide position in solvent-exposed α-helical regions.
- R1p simplifies PRE data interpretation by eliminating the need to account for spin label flexibility.
- R1p is a valuable tool for accurate biomolecular distance measurements and studying transient states.
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