Speaker
Jan Skov Pedersen
(1Department of Chemistry, Aarhus University, Aarhus, Denmark)
Description
Jan Skov Pedersen1,2, Jannik N. Pedersen1,2, Jeppe Lyngsø1,2, Jørn Døvling Kaspersen1,2, Anne Søndergaard1,2, Daniel Jhaf Madsen2, T. Zinn3, T. Narayanan3, and Daniel E. Otzen2
1Department of Chemistry, Aarhus University, Aarhus, Denmark
2Interdisciplinary Nanoscience center (iNANO), Aarhus University, Aarhus, Denmark
3European Synchrotron Radiation Facility, Grenoble, France
email: jsp@chem.au.dk (J.S.P.)
The strong and usually denaturing interaction between anionic surfactants (AS) and proteins/enzymes has both benefits and drawbacks: For example, it is in good use in electrophoretic mass determinations (SDS-PAGE) but limits enzyme efficiency in detergent formulations. Therefore, studies of the interactions between proteins and AS as well as non-ionic surfactants (NIS) are of both basic and applied relevance. The AS sodium dodecyl sulfate (SDS) denatures and unfolds globular proteins under most conditions. In contrast, it has been shown that the NIS octaethylene glycol monododecyl ether (C12E8) protects bovine serum albumin (BSA) from unfolding in SDS. We have shown recently that globular proteins unfolded by SDS can be refolded upon addition of C12E8. Four proteins, BSA, α-lactalbumin, (αLA), lysozyme (LYZ), and β-lactoglobulin (βLG), were studied by small-angle X-ray scattering (SAXS) and both near- and far-UV circular dichroism (CD). All proteins form complexes with SDS with a structural organization as protein-decorated micelles, in which the protein preserves secondary structure. For βLG, there is a quite spectacular transition from mainly -sheet structure in the native protein to mainly -helical structure in the complexes. All proteins were attempted refolded by the addition of C12E8. Except for apo αLA, which has a molten globular state, the proteins did not interact with C12E8 alone. The addition of C12E8 to the protein-SDS samples resulted, except for aLA, in refolding of the tested proteins and dissociation from surfactant micelles. It was concluded that C12E8 competes with globular proteins for association with SDS, making it possible to release and refold SDS-denatured proteins by adding sufficient amounts of C12E8. The last part of the talk will describe recent work using synchrotron radiation SAXS in combination with stopped-flow techniques on the kinetics of unfolding and refolding with emphasis on βLG. For this protein, the preliminary analysis shows a fast aggregation, when SDS is added, and a gradual conversion of the structure to highly symmetric protein-decorated micelle structures that is nearly complete in 10 s. The refolding is significantly slower with time constants of minutes, however, CD and Trp fluorescence in our home lab reveal that there is also a much faster process that is not captured in the SAXS measurements, which is a secondary structure conversion. In the work, both simple analysis in terms of measured basis functions (SAXS data) as well as modelling on absolute scale have been used and will be briefly described.
Kaspersen JD, Søndergaard A, Madsen DJ, Otzen DE, Pedersen JS. Refolding of SDS-Unfolded Proteins by Nonionic Surfactants. Biophys J. 2017 112(8):1609-1620.
Primary author
Jan Skov Pedersen
(1Department of Chemistry, Aarhus University, Aarhus, Denmark)
