Effects of macromolecular crowding on folded protein homologs: global versus local consequences
(English)Manuscript (preprint) (Other academic)
Proteins function in cellular environments that are crowded with other biomolecules. This reduction of available space may change biophysical properties of proteins as compared to dilute solutions in vitro. Here we have investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. First, as expected for an excluded volume effect, the thermal stability increased for S16Meso in the presence of dextran 20. In accord with a dominating entropic effect, chemical denaturation experiments at different fixed temperatures showed that the crowding effect was temperature independent. Far-UV circular dichroism spectra did not reveal any secondary structural changes in the folded proteins in presence of dextran 20. However, Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY in S16Meso depend on the level of crowding agent. The BODIPY-group was attached at three specific positions in S16Meso. All S16Meso variants exhibited a decrease in the average distance up to 100 mg/mL dextran 20, indicating folded-state compaction, whereas the change in distance became anisotropic at higher dextran concentrations. In contrast, the two S16Thermo mutants, that are thermodynamically more stable than the mesophilic variants, did not show any change in distance upon increasing dextran 20 concentrations. Notably, the BODIPY fluorescence quantum yields and lifetimes of the two homologs decreased gradually in the presence of dextran 20. To investigate the origin of this, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labelled dextran. The results reveal quenching between tyrosine and BODIPY at all concentrations of Tyr-dextran. The data also suggests that dynamic local interactions between protein and crowding agent can occur at some conditions.
Excluded volume, Fluorescence, Tryptophan-BODIPY, Förster resonance energy transfer, dextran 20, small ribosomal protein S16
Research subject Physical Chemistry
IdentifiersURN: urn:nbn:se:umu:diva-86791OAI: oai:DiVA.org:umu-86791DiVA: diva2:704065
FunderSwedish Research Council, 550021104