Heat Capacities and a Snapshot of the Energy Landscape in Protein GB1 from the Pre-denaturation Temperature Dependence of Backbone NH Nanosecond Fluctuations

Author： Idiyatullin D. Nesmelova I. Daragan V.A. Mayo K.H.

Publisher： Academic Press

ISSN： 0022-2836

Source： Journal of Molecular Biology, Vol.325, Iss.1, 2003-01, pp. : 149-162

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Abstract

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 backbone¹⁵NH 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; theF(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.© 2002 Elsevier Science Ltd