Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
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SHR Neuro Krebs Kardio Lipid Stoffw Microb
Alderson, TR; Charlier, C; Torchia, DA; Anfinrud, P; Bax, A.
Monitoring Hydrogen Exchange During Protein Folding by Fast Pressure Jump NMR Spectroscopy.
J Am Chem Soc. 2017; 139(32): 11036-11039.
Doi: 10.1021/jacs.7b06676
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- Führende Autor*innen der Med Uni Graz
- Alderson Thomas Reid
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- Abstract:
- A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca. 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca. 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the β1-β2 hairpin, previously shown to be already lowly populated in the urea-denatured state.
- Find related publications in this database (using NLM MeSH Indexing)
- Humans - administration & dosage
- Hydrogen - chemistry
- Hydrostatic Pressure - administration & dosage
- Models, Molecular - administration & dosage
- Nuclear Magnetic Resonance, Biomolecular - methods
- Point Mutation - administration & dosage
- Protein Conformation - administration & dosage
- Protein Denaturation - administration & dosage
- Protein Folding - administration & dosage
- Protein Structure, Secondary - administration & dosage
- Ubiquitin - chemistry, genetics