Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro.
One of the central problems in protein folding is how amino acid sequences within polypeptide chains direct polypeptide chain folding and avoid off-pathway aggregation both in intracellular environments and in the test tube. The tailspike protein of phage P22 is a model system for which genetic analysis has permitted mutational dissection of the role of amino acid positions in the polypeptide chain in directing its in vivo folding. Two classes of mutations that affect intracellular folding and aggregation have been characterized; temperature-sensitive folding (tsf) mutants and second-site suppressors of tsf mutants. Here we report the effects of these mutations on the in vitro refolding and aggregation pathway of the purified proteins. The tsf mutations reduced refolding yields at high temperature and increased aggregation, while second-site suppressors enhanced refolding and inhibited aggregation in the test tube. For both types of mutations, the strength of the effects observed in vitro correlated with their in vivo phenotypes. The results confirm that the mutations act intrinsically on the folding pathway of the tailspike polypeptide and not through accessory proteins.
Mitraki
A
A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Danner
M
M
King
J
J
Seckler
R
R
D000595:Amino Acid Sequence; D017100:Bacteriophage P22 / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017376:Viral Tail Proteins
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014764:Viral Proteins; D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage