Abstract
To investigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro, we constructed a set of variants of the N-terminal domain of lambda repressor with a wide range of melting temperatures. Pulse-chase experiments showed that, within this set, the proteins that are most thermally stable have the longest intracellular half-lives and vice versa. Moreover, second-site mutations which act directly or indirectly to increase the thermodynamic stability of the native N-terminal domain were found to suppress the intracellular degradation of one of the unstable mutants. These data suggest that thermal stability is, indeed, a key determinant of the proteolytic susceptibility of this protein in the cell. It is not the sole determinant, however, as sequences at the extreme C terminus of the N-terminal domain can influence proteolytic sensitivity without affecting the stability of the native structure. We propose that the thermal stability of the N-terminal domain of lambda repressor is an important determinant of its proteolytic sensitivity because degradation proceeds primarily from the unfolded form and that sequence determinants within the unfolded chain influence whether the unfolded protein will be a good substrate for proteolytic enzymes.
Highlights
TOinvestigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro,we constructed a set of variants of the N-terminal domain of X repressor with a wide range of meIting temperatures
We propose a bipartite model for protein turnover: the thermodynamic stabilitoyf a protein determines the fractionof the protein thatwill be in an unfolded and proteolyticallysusceptible state, while sequence determinants within uthnefolded protein chain determine how effectively it will act as a substrate for proteolytic enzymes
The main features of the model are as follows: (i) the denatured or unfolded form of the protein is assumed to be the only form of the protein that is a substrate for proteolysis
Summary
TOinvestigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro,we constructed a set of variants of the N-terminal domain of X repressor with a wide range of meIting temperatures. Secondsite mutations which act directly or indirectly to increase the thermodynamic stability of the native Nterminal domain were found to suppress the intracellular degradation of one of theunstable mutants. These data suggest that thermal stability is, a key determinant of the proteolytic susceptibility ofthis protein in the cell. It is not the soledeterminant, as sequences at the extremeC terminus of the Nterminal domain can influence proteolytic sensitivity without affecting the stabilityof the native structure. We propose a bipartite model for protein turnover: the thermodynamic stabilitoyf a protein determines the fractionof the protein thatwill be in an unfolded and proteolyticallysusceptible state, while sequence determinants within uthnefolded protein chain determine how effectively it will act as a substrate for proteolytic enzymes
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