Postdoctoral Fellow University of Arkansas West Fork, Arkansas, United States
Mercede Furr (University of Arkansas)| Sreenivasulu Basha (University of Arkansas)| Shilpi Agrawal (University of Arkansas)| Zeina Alraawi (University of Arkansas)| Piyasi Ghosh (University of Arkansas)| T.K.S. Kumar (University of Arkansas)| Ruben Ceballos (University of Arkansas)
Group II chaperonins are the most abundant proteins in hyperthermophilic archaea of the order Sulfolobales. These “heat shock” proteins (HSPs) consist of three subtypes: HSPα, HSPβ, and HSPγ. Expression of HSPα and HSPβ is upregulated in response to thermal shock and these subunits form nonameric double-ring structures (18-mers) referred to as HSP complexes. HSPα and HSPβ subunits form complexes as homooligomers or heterooligomers at different combination ratios. The natural role of these chaperonin complexes is to protect other cellular proteins (e.g., enzymes) from damage during heat shock. In an effort to develop a thermotolerant enzyme binding platform for industrial and/or agricultural applications, an engineered fusion protein was developed by combining a circular permutant of HSPβ from Sulfolobus shibatae with Type 1 cohesin from the cellulolytic bacterium, Clostridium thermocellum with the idea of developing a thermotolerant “artificial” cellulosome. In this study, the relative thermal stability and acido-stability of the natural and engineered HSP subunits was assessed using biophysical methods. Intrinsic tryptophan fluorescence, differential scanning calorimetry (DSC), and far UV circular dichroism spectroscopy suggest that the native structure of HSPα and HSPβ is not significantly perturbed at neutral pH over a temperature range of 25-90⁰C. The engineered HSPβ-coh fusion protein is slightly less thermally stable and is prone to aggregation past 85⁰C when compared to the natural HSPs. All subunits show shifts in structural conformation under low pH conditions (pH 2 and 3). The results of limited trypsin digestion show that the backbone structure of HSPβ is the least flexible followed closely by HSPα. HSPβ-coh exhibited the highest backbone flexibility. All subunits appear to be more compact at neutral pH and higher temperatures (75⁰C, 85⁰C, and 90⁰C) as lower relative fluorescence intensity (RFI) upon ANS binding is observed under these conditions as compared to the RFI of the subunits at pH2 and 25⁰C. The observations regarding the increased flexibility and lowered thermal stability of HSPβ-coh is due to the added cohesin moiety. This is consistent with the conclusions drawn from intrinsic fluorescence and ANS binding data. Perturbations in secondary and tertiary structures are observed for all subunits at pH <3. However, the exact nature of these structural changes remains unknown. At pH <3, it appears that HSP structures are less compact while still avoiding aggregation. Overall, the HSP subunits exhibit high thermal stability but undergo structural changes in low pH conditions.