Obtaining Hydrodynamic Radii of Intrinsically Disordered Protein Ensembles by Pulsed Field Gradient NMR Measurements
| Autor: | Sarah Leeb, Jens Danielsson |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Quantitative Biology::Biomolecules
0303 health sciences Materials science Hydrodynamic radius A protein Magnetic field gradient 010402 general chemistry 01 natural sciences 0104 chemical sciences 03 medical and health sciences Chemical physics Pulsed field gradient Spectroscopy 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071605233 |
| Popis: | In the disordered state, a protein exhibits a high degree of structural freedom, in both space and time. For an ensemble of disordered or unfolded proteins, this means that the ensemble comprises a high diversity of structures, ranging from compact collapsed states to fully extended polypeptide chains. In addition, each chain is highly dynamic and undergoes conformational changes and local dynamics on both fast and slow timescales. The size properties of disordered proteins are thus best described as ensemble averages. A straightforward measure of the size is the hydrodynamic radius, RH, of the ensemble. Since the disordered state is conformationally fluid, the observed RH does not refer to a particular shape or fold. Instead, it should be interpreted as a measure for the average compaction of the structural ensemble. In addition to characterizing the disordered ensemble itself, RH can be used to, with good precision, monitor changes in the ensemble size properties upon functional interactions of the disordered protein, e.g., dimerization, ligand binding, and folding pathways. Here, we present a step-by-step protocol for diffusion measurements using pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. We describe how to calibrate the magnetic field gradient and offer different schemes for sample preparation. Finally, we describe how to obtain RH directly from the diffusion coefficient as well as from using an internal standard as a reference. |
| Databáze: | OpenAIRE |
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