Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein.
Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.
Sturtevant
J M
JM
Department of Chemistry, Yale University, New Haven, Connecticut 06511.
Yu
M H
MH
Haase-Pettingell
C
C
King
J
J
D000596:Amino Acids; D009154:Mutation; D011487:Protein Conformation; D012483:Salmonella Phages / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D017376:Viral Tail Proteins
D000596:Amino Acids; D014764:Viral Proteins; D017376:Viral Tail Proteins