An essential proline in lambda repressor is required for resistance to intracellular proteolysis.
Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, J.F., & Sauer, R.T. (1990) Proteins: Struct., Funct., Genet. (in press)]. To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases. The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Reidhaar-Olson
J F
JF
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Parsell
D A
DA
Sauer
R T
RT
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000235:genetics*; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013237:Stereoisomerism
D004269:DNA, Bacterial; D012097:Repressor Proteins; D011392:Proline; D010450:Endopeptidases