Abstract
The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 coat protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of coat protein. Additional amino acid residues that are critical to the folding process of P22 coat protein were identified by isolating second site suppressors of the tsf coat proteins. Suppressor substitutions isolated from the phage carrying the tsf coat protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf coat protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the coat protein sequence and were 8-190 amino acid residues away from the tsf parent. Although the folding of coat proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of coat protein.
Highlights
Sequence controls both folding and misfolding will allow us to understand how these diseases and other processes related to misfolding occur
If the tsf amino acid substitutions do not affect the folding in the same way, it would be unlikely that the same suppressor sites would be isolated for several tsf coat protein mutants; instead, site-specific suppressors of tsf coat protein mutants would be isolated
Suppressors of 18 tsf coat protein mutants have been isolated in order to determine the positions of additional amino acids crucial to stabilizing the folding intermediate(s) affected by the tsf amino acid substitutions
Summary
A search of second site suppressors has been utilized to determine gene product interactions [22,23,24,25,26] In this case, a survey of second site suppressors of coat proteins with tsf amino acid substitutions could be expected to reveal interactions between residues in the affected folding intermediate, and not with proteins that interact with the tsf coat protein because the folding of coat protein into monomeric units normally proceeds without the interaction with other phage proteins [27]. If all the tsf substitutions affect the same intermediate in a folding pathway, a set of suppressor amino acid substitutions that stabilize the intermediate might be expected to be repeatedly isolated from several tsf coat protein mutants. The global suppressors identify a region critical to proper folding of coat protein
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