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Backbone dynamics of barstar: a (15)N NMR relaxation study
S C Sahu1, A K Bhuyan, A Majumdar
1Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India.
Proteins
|October 31, 2000
Summary
This study reveals barstar protein dynamics using nuclear magnetic resonance (NMR) spectroscopy. Backbone dynamics and molecular motions were characterized, providing insights into protein flexibility and conformational averaging.
Area of Science:
- Biophysics
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Understanding protein dynamics is crucial for elucidating protein function.
- Barstar, a protein inhibitor of barnase, serves as a model system for studying protein folding and stability.
Purpose of the Study:
- To investigate the backbone dynamics of uniformly (15)N-labeled barstar using (15)N relaxation data.
- To determine molecular parameters such as rotational correlation time, order parameters, and internal motion correlation times.
- To analyze conformational averaging motions in barstar.
Main Methods:
- Proton-detected 2D (1)H-(15)N NMR spectroscopy was employed at 32°C and pH 6.7.
- (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and heteronuclear (1)H-(15)N NOEs were measured.
- Model-free formalism and reduced spectral density mapping were used to analyze relaxation data.
Main Results:
- The overall rotational correlation time (tau(m)) was determined to be 5.2 ns (model-free) and 5.7 ns (spectral density mapping).
- Generalized order parameters (S(2)) ranged from 0.68 to 0.98, with an average of 0.85 ± 0.02, indicating significant internal flexibility.
- Conformational averaging motions, analyzed via exchange broadening (R(ex)), were identified for a small subset of residues.
Conclusions:
- The determined rotational correlation time differs from previous fluorescence data, highlighting the importance of NMR relaxation studies.
- The observed order parameters suggest considerable backbone flexibility, but show limited correlation with X-ray B-factors.
- NMR relaxation data provides valuable insights into the dynamic behavior and conformational landscape of barstar.
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