Thermostability of Temperature-sensitive Folding Mutants of the P22 Tailspike Protein

J M Sturtevant,M H Yu,C Haase-Pettingell,J King

doi:10.1016/s0021-9258(18)81678-7

Abstract

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.

Highlights

Because the folding defects were observed in vivo, gated forms of the polypeptide chain often accumulate (Jaenicke, 1987)
The initial successes in refolding denatured proteins back to thenative state i n vitro supported the view that thefolding of the polypeptide chain into the native conformation was thermodynamically determined, a consequenceof the minimal energy of the native state (Anfinsen, 1973; Anfinsen and Scheraga, 1975; Creighton, 1984;Baldwin and Eisenberg, equated with effects on folding (Shortle and Meeker, 1986; Becktel and Schellman, 1987;Schellman, 1987).For two-state systems the mutants used to study folding processes have been initially selected on the basis of their destabilization of the native state (Hecht et al, 1984; Matsumura et al, 1986; Alber et al.,1987)
Thermostability of Native Tailspikes in Vitro-Seventsf mutant proteins would be less stable than the native stateof mutant proteins were chosen forcalorimetry

Summary

Cholnr Arrocloted but not fully folded

The tsf mutants carrysingle amino acid substitutions which interfere with the intracellular chainfolding and association pathway at the high end of the temperature range for phage growth (Smith and King,1981; Goldenberg et al, 1983; King et al, 1987). Mutant polypeptide mutation, amNl14, in gene 5 prevents the synthesis of the major coat protein In such cells the tailspikes accumulate as soluble species, and, due to thefailure to package the DNA, synthesis continuesat a very high level. Preparation of Proteins-The purification of the protein samples chains released from the ribosome at restrictive temperature used for these experiments is described by King and Yu (1986) and (38-40 "C) fail to reach the native state (Smith and King, 1981).

RESULTS

MATERIALS ANDMETHODS

Protein t*

DISCUSSION

All of the tsf mutant proteins were stable to temperatures