GENETIC ENGINEERING TECHNOLOGY HAS NOW ADVANCED TO THE POINT WHERE IT IS IN PRINCIPLE POSSIBLE TO CLONE THE GENE FOR ANY DESIRED PROTEIN AND TO SYNTHESIZE IT IN QUANTITY IN BACTERIA. THUS THE TECHNOLOGY IS AVAILABLE NOT ONLY TO ENABLE KNOWN ENZYMES OR PROTEINS TO BE PRODUCED ON AN INDUSTRIAL SCALE, BUT ALSO IT IS POSSIBLE TO SYNTHESIZE PROTEINS OF ANY DESIRED SEQUENCE. BEFORE THE POINT IS REACHED WHERE NEW ENZYMES CAN BE DESIGNED AND SYNTHESIZED DE NOVO TO CATALYSE INDUSTRIAL PROCESSES, MUCH MORE MUST BE LEARNT ABOUT THE FORCES CONTROLLING THE FOLDING AND ASSEMBLY OF PROTEINS INTO SPECIFIC THREE-DIMENSIONAL STRUCTURES.
Recurrent tertiary motifs represent solutions to basic structural requirements for protein stability. Understanding them well would facilitate the engineering of proteins with novel functions. Structural simplicity and functional diversity make the 4-alpha-helix bundle motif a prime candidate for protein design. The regularity, stability and functional properties of the 2 proteins studied make them ideal model systems for the study of the interactions determining folding and assembly of 4-alpha-helix bundles.

Examination of the effects of amino acid substitutions on ROP and ferritin could lead to deeper insight into the folding, subunit assembly and stability of the 4-alpha-helical proteins. In a combined theoretical and experimental approach, molecular graphics were used in the design of mutants which were then constructed and characterized using biochemical, molecular, genetic and immunological approaches. Some mutants were crystallized and their structures were studied at high resolution. The results of procedures developed for the study of folding, assembly and function, protein modelling work and microcalorimetry, formed a database documenting basic requirements for protein folding, stability and function.

ROP mutants with altered turns and cores were selected and characterized crystallographically. The ROP structure tolerates considerable loop changes and its folding is entirely dominated by the hydrophobic core interactions. While cavities in the interior of the protein have generally destabilizing effects, there are striking exceptions which are correlated with the hydration of the core. In ferritin, the loop between helices D and E is less tolerant to changes and the folding decision of the E helix is taken upon assembly. The ferroxidase activity is in the H chain, in the interior of the 4-helix bundle. Further metal binding sites were found along the symmetry axes of the molecule, which appear also to be important for stability. Both proteins are exc ellent scaffolds for the attachment of binding sites of industrial interest.
THE SPECIFIC AIM OF THIS PART OF THE PROJECT IS TO DEVELOP A DATABASE WHICH WILL DOCUMENT IN TERMS OF STRUCTURAL CHANGES AND STABILITY THE EFFECTS RESULTING FROM MUTATIONS OF THE ROP PROTEINS. THIS PROTEIN FUNCTIONS IN THE CONTROL OF PLASMID COPY NUMBER IN E. COLI. THE INFORMATION THUS OBTAINED WILL BE INCORPORATED IN CURRENTLY USED TOOLS FOR PROTEIN DESIGN. WITH THIS TOOLS IT SHALL BE ATTEMPTED TO DESIGN PROTEINS CARRYING A DESIRED ENZYMATIC FUNCTION. THE DESIRED MUTANTS WILL BE DESIGNED USING EXISTING COMPUTER TOOLS FOR PROTEIN DESIGN AND A MOLECULAR GRAPHICS DEVICE. THE STRUCTURAL AND THERMOSTABILITY PROPERTIES OF THE MUTANTS WILL BE USED TO CREATE THE DATABASE. FINALLY, THE APPLICANT WILL ATTEMPT TO IMPROVE EXISTING ALGORITHMS FOR PROTEIN FOLDING AND TRY TO DEVELOP NEW CONCEPTS APPLICABLE IN THE FIELD OF PROTEIN DESIGN AND ENGINEERING.