Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone 15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5-50°C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the F(ω) = 2ωJ(ω) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33 kJ/mol residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most "rigid" segments of the fold: β-strands 1 and 4 and the α-helix. Trends in this landscape also parallel the free energy of folding-unfolding derived from hydrogen-deuterium (H-D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H-D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100 J/mol K residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.
Bibliographical noteFunding Information:
This work was supported by a research grant from the National Institutes of Health (NIH, GM-58005) and benefited from use of the high field NMR facility at the University of Minnesota. We would like to thank Judy Haseman for preparing 15 N-enriched samples of protein GB1.
- Correlation times
- Motional models
- N relaxation
- Spectral density