Abstract
Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contrast variation (H2O/D2O exchange) allows focussing selectively on the signal of specific proteins in multi-protein complexes or mixtures of isolated proteins. We illustrate this unique capacity by the example of a functional protein-degradation system in solution, the PAN-20S proteasome complex in the presence of a protein substrate, ssrA-tagged GFP. By comparing experimental SANS data with synthetic SAXS (small-angle X-ray scattering) data, predicted for the same system under identical conditions, we show that SANS, when combined with macromolecular deuteration and solvent contrast variation, can specifically focus on the conformation of the PAN unfoldase, even in the presence of very large GFP aggregates. Likewise, structural information of native GFP states can be visualized in detail, even in the presence of the much larger PAN-20S unfoldase-protease oligomers, which would dominate the overall scattering signal when using X-rays instead of neutrons.
Highlights
Small-angle X-ray (SAXS) and neutron (SANS) scattering provide structural information on biomacromolecules in solution on the nanometre length scale [1]
SAXS cannot distinguish between different proteins in solution since they have, in general, a comparable average electron density and contrast with the solvent, i.e. their intrinsic electron density cannot be chemically modified in a global and homogeneous way
By using selective per-deuteration of either PAN or Green Fluorescent Protein (GFP) in a 42% D2O buffer, we were able to focus selectively on either of both proteins during the active degradation process: under these labelling and solvent contrast conditions, the signal of the hydrogenated protein species in a reaction mixture are completely masked and the perdeuterated one has a strong contrast (Fig. 1). We demonstrate that this is even true if the hydrogenated species or oligomerization states are several orders of magnitude bigger than the deuterated ones
Summary
Small-angle X-ray (SAXS) and neutron (SANS) scattering provide structural information on biomacromolecules in solution on the nanometre length scale [1]. By using selective per-deuteration (i.e. full deuteration) of either PAN or GFP in a 42% D2O buffer, we were able to focus selectively on either of both proteins during the active degradation process: under these labelling and solvent contrast conditions, the signal of the hydrogenated protein species in a reaction mixture are completely masked and the perdeuterated one has a strong contrast (Fig. 1). We demonstrate that this is even true if the hydrogenated species or oligomerization states are several orders of magnitude bigger than the deuterated ones. By thermo-activation at 55 °C, the protein degradation reactions were followed at a time-resolution of 30 seconds during 45 minutes, coupled with online fluorescence spectroscopy [8]
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