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Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9.
J Mol Biol. 1998 Dec 18; 284(5):1661-70.JM

Abstract

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

Authors+Show Affiliations

Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY, 11794-3400, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

9878377

Citation

Kuhlman, B, et al. "Global Analysis of the Effects of Temperature and Denaturant On the Folding and Unfolding Kinetics of the N-terminal Domain of the Protein L9." Journal of Molecular Biology, vol. 284, no. 5, 1998, pp. 1661-70.
Kuhlman B, Luisi DL, Evans PA, et al. Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9. J Mol Biol. 1998;284(5):1661-70.
Kuhlman, B., Luisi, D. L., Evans, P. A., & Raleigh, D. P. (1998). Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9. Journal of Molecular Biology, 284(5), 1661-70.
Kuhlman B, et al. Global Analysis of the Effects of Temperature and Denaturant On the Folding and Unfolding Kinetics of the N-terminal Domain of the Protein L9. J Mol Biol. 1998 Dec 18;284(5):1661-70. PubMed PMID: 9878377.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9. AU - Kuhlman,B, AU - Luisi,D L, AU - Evans,P A, AU - Raleigh,D P, PY - 1999/1/8/pubmed PY - 1999/1/8/medline PY - 1999/1/8/entrez SP - 1661 EP - 70 JF - Journal of molecular biology JO - J Mol Biol VL - 284 IS - 5 N2 - The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/9878377/Global_analysis_of_the_effects_of_temperature_and_denaturant_on_the_folding_and_unfolding_kinetics_of_the_N_terminal_domain_of_the_protein_L9_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(98)92246-X DB - PRIME DP - Unbound Medicine ER -