Some of the amino acid interactions critical for determining three dimensional protein structure must somehow direct the conformation of folding intermediates. The thermostable tailspike endorhamnosidase of phage P22 has a thermolabile early intermediate in its folding and subunit association pathway. Over 100 temperature sensitive folding mutations have been isolated which either further destabilize the thermolabile intermediate, or otherwise alter early kinetic steps. Many of the substitutions of glycines and hydrophilic residues are at the surface of the native structure, probably associated with surface turns. Since tailspike secondary structure is over 50% beta-sheet/turn, these are probably beta turns, and the mutations identify residues influencing their formation. The specific objectives of the award are to: 1) Identify the sites and amino acid substitutions for the remainder of the tsf mutations; 2) For those substitutions altering charge, determine which are at the surface and therefore turn candidates, and analyze the folding defect in these mutants; 3) Isolate second site suppressors of these defects, to determine whether the interacting residues are local or distant in the chain; 4) Isolate and characterize the protrimer intermediate from wild type and mutant infected cells; 5) Isolate and characterize the aggregated chains that accumulate at high temperature as a means of investigating the conformation of the early intermediate from which it forms; 6) Purify tsf mutant and tsf/suppressor-tsf double mutant proteins for collaborative X-ray diffraction, Raman spectroscopy, in vitro refolding, and differential scanning calorimetry studies.
