title: High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.
abstract: Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.
authors: Department of Chemistry & Chemical Biology, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Yi
authors: Y
authors: Zhang
authors: Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Soichiro
authors: S
authors: Kitazawa
authors: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794, United States.
authors: Ivan
authors: I
authors: Peran
authors: Graduate Program in Biochemistry and Structural Biology, Stony Brook University , Stony Brook, New York 11794, United States.
authors: Natalie
authors: N
authors: Stenzoski
authors: NMR Core Facility, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Scott A
authors: SA
authors: McCallum
authors: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794, United States.
authors: Daniel P
authors: DP
authors: Raleigh
authors: Department of Chemistry & Chemical Biology, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.|Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Catherine A
authors: CA
authors: Royer
mesh_terms: D001411:Geobacillus stearothermophilus / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011312:Pressure; D011487:Protein Conformation; D000072417:Protein Domains; D017510:Protein Folding; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D012269:Ribosomal Proteins