In vivo and in vitro function of GroEL mutants with impaired allosteric properties.
Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C. This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.
Fridmann
Y
Y
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Ulitzur
S
S
Horovitz
A
A
D000494:Allosteric Regulation; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D009154:Mutation; D011489:Protein Denaturation; D014733:Vibrio / Q000201:enzymology
D018834:Chaperonin 60; D017931:DNA Primers; D008156:Luciferases