To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (DeltaCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low DeltaCp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H. Study holds ProTherm entries: 13061, 13062, 13063, 13064, 13065, 13066 Extra Details: thermodynamic stability of proteins; chimera; stability curves; heat capacity; folding core; ribonuclease H
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||12|
|Proteins||Ribonuclease H ; Ribonuclease H ; Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: m prot_conc:50 micro g/mL ; Experimental Assay: dG_H2O prot_conc:50 micro g/mL ; Experimental Assay: m prot_conc:50 micro g/mL ; Experimental Assay: dG_H2O prot_conc:50 micro g/mL ; Experimental Assay: m prot_conc:50 mg/mL ; Experimental Assay: dG_H2O prot_conc:50 mg/mL ; Experimental Assay: dCp ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MNPSPRKRVALFTDGACLGNPGPGGWAALLRFHAHEKLLSGGEACTTNNRMELKAAIEGLKALKEPCEVDLYTDSHYLKKAFTEGWLEGWRKRGWRTAEGKPVKNRDLWEALLLAMAPHRVRFHFVKGHTGHPENERVDREARRQAQSQAKTPCPPRAPTLFHEEA ; Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|