DEVELOPMENT OF RULES FOR FOLDING OF BIOTECHNOLOGY PRODUCED PROTEIN

IN GENERAL, PROTEINS FOLD WITH HYDROPHOBIC RESIDUES BURIED, AWAY FROM WATER. REVERSIBLE PROTEIN FOLDING DUE TO HYDROPHOBIC INTERACTIONS RESULTS FROM INVERSE TEMPERATURE TRANSITIONS WHERE FOLDING OCCURS ON RAISING THE TEMPERATURE. SINCE WARM-BLOODED ANIMALS PROVIDE AN INFINITE HEAT RESERVOIR, THE TRANSITION TEMPERATURE, TT, NOT THE ENDOTHERMIC HEAT, DETERMINES THE FOLDED STATE AT PHYSIOLOGICAL TEMPERATURES. AS THE TEMPERATURE IS RAISED THROUGH THE INVERSE TEMPERATURE TRANSITION, THE POLYPEPTIDE OR PROTEIN FOLDS BY HYDROPHOBIC INTERACTIONS INTO A MORE-ORDERED STATE. THE TEMPERATURE OF THE INVERSE TEMPERATURE TRANSITION, TT, CAN BE SHIFTED BY AN EXTERNAL VARIABLES; THEREFORE IT BECOMES POSSIBLE, ISOTHERMALLY, TO DRIVE THE FOLDING/UNFOLDING TRANSITION: (1) BY A CHANGE IN THE MEAN HYDROPHOBICITY OF THE POLYPEPTIDE (E.G., BY PROTONATION/DEPROTONATION OF A FUNCTIONAL SIDE CHAIN, BY ENZYMATIC PHOSPHORYLATION/DEPHOSPHORYLATION OF A SERINE MOIETY, ETC.), (2) BY A CHANGE IN THE SOLVENT COMPOSITION, (3) BY A CHANGE IN PRESSURE, AND, (4) BY A CHANGE IN OXIDATIVE STATE OF A PROSTHETIC GROUP. THE QUANTITATION OF THE CONTRIBUTION OF EACH AMINO ACID TO THE TEMPERATURE OF THE INVERSE TEMPERATURE TRANSITION RESULTED IN A NEW HYDROPHOBICITY SCALE BASED ON THE VALUES OF TT FOR EACH AMINO ACID RESIDUE AS A GUEST IN A NATURAL REPEATING PEPTIDE SEQUENCE, THE HIGH POLYMERS OF WHICH EXHIBIT REVERSIBLE INVERSE TEMPERATURE TRANSITIONS. THE TRANSITION TEMPERATURE SCALE CAN SUCCESSFULLY PREDICT TRANSMEMBRANE HELICES FOR MEMBRANE PROTEINS. TO DEVELOP FURTHER RULES AND METHODOLOGIES FOR EXPERIMENTALLY DIRECTING PROTEIN FOLDING, IT IS NECESSARY SIMILARLY TO QUANTITATE THE EFFECTS OF A RANGE OF SOLVENT COMPOSITIONS ON SHIFTING THE VALUE OF TT FOR EACH RESIDUE, TO DETERMINE DISTANCE EFFECTS OF A RANGE OF SOLVENT COMPOSITIONS ON SHIFTING THE VALUE OF TT FOR EACH RESIDUE, TO DETERMINE DISTANCE EFFECTS IN HYDROPHOBICITY INDUCED PKA SHIFTS, AND TO DETERMINE HOW LARGE AN HYDROPHOBIC DOMAIN IS REQUIRED BEFORE IT CAN FOLD INDEPENDENTLY. THE OBJECTIVES OF THE SBIR PHASE I ARE TO PROVIDE ADDITIONAL INFORMATION ON EACH OF THESE NEW QUANTITATIONS WITH THE PURPOSE OF DEVELOPING RULES FOR CHAPERONING PROTEIN FOLDING WHICH CAN BE GENERAL AND