title: Analyzing protein folding cooperativity by differential scanning calorimetry and NMR spectroscopy.
abstract: Some marginally stable proteins undergo microsecond time scale folding reactions that involve significant populations of partly ordered forms, making it difficult to discern individual steps in their folding pathways. It has been suggested that many of these proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) relaxation dispersion experiments, and an analysis of the temperature dependence of amide (1)H and (15)N chemical shifts. We applied this method to the PBX homeodomain (PBX-HD), which folds on the microsecond time scale and produces a broad DSC thermogram with an elevated and steeply sloping native-state heat capacity baseline, making it a candidate for barrierless folding. However, by globally fitting the NMR thermal melt and DSC data, and by comparing these results to those obtained from the NMR relaxation dispersion experiments, we show that the native form of the protein undergoes two-state exchange with a small population of the thermally denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms and firmly establishes that folding of PBX-HD is cooperative. Further, we see evidence of large-scale structural and dynamical changes within the native state by NMR, which helps to explain the broad and shallow DSC profile. This study illustrates the potential of combining calorimetry with NMR dynamics experiments to dissect mechanisms of protein folding.
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6.
authors.forename: Patrick
authors.initials: P
authors.lastname: Farber
authors.forename: Hariyanto
authors.initials: H
authors.lastname: Darmawan
authors.forename: Tara
authors.initials: T
authors.lastname: Sprules
authors.forename: Anthony
authors.initials: A
authors.lastname: Mittermaier
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D018398:Homeodomain Proteins / Q000737:chemistry; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding
chemical_list: D018398:Homeodomain Proteins