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Content archived on 2024-04-15

MODEL SYSTEMS FOR PRODUCTION FOLDING AND ASSEMBLY OF STABLE PROTEINS OVEREXPRESSED IN E.COLI

Objective

GENETIC ENGINEERING TECHNOLOGY HAS TO ADVANCED TO THE POINT WHERE IT IS IN PRINCIPLE POSSIBLE TO CLONE THE GENE FOR ANY DESIRED PROTEIN AND TO SYNTHESIS 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 PRINCIPAL AIM OF THIS PART OF THE PROJECT IS TO ARRIVE AT A BETTER UNDERSTANDING OF FACTORS CONTROLLING THE SUBUNIT FOLDING, SUBUNIT ASSOCIATION, MOLECULAR STABILITY, MOLECULAR FUNCTION, ANTIGENICITY AND CRYSTALLISABILITY OF THE IRON-STORAGE PROTEIN FERRITIN. NATURAL FERRITIN IS COMPOSED OF 24 SUBUNITS OF TWO DIFFERENT TYPES KNOWN AS H AND L IN VARYING PROPORTIONS. THE FIRST APPROACH WILL BE TO STUDY THE STRUCTURE AND PROPERTIES OF FERRITIN MOLECULES COMPOSED SOLELY OF EITHER H OR L SUBUNITS. AFTERWARDS THE EFFECTS ON THE STRUCTURE AND PROPERTIES CAUSED BY SPECIFIC, SITE-DIRECTED CHANGES IN THE AMINO ACID SEQUENCE OF H OR L SUBUNITS WILL BE INVESTIGATED. IN THE LAST PART STRUCTURAL COMPARISONS OF FERRITIN AND ROP PROTEIN AND THEIR MUTANTS WILL BE MADE SO AS TO ANALYSE THE EXTENT TO WHICH THESE PROTEINS HAVE COMMON FEATURES IN THEIR SUBUNIT FOLDING PATTERNS.

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Coordinator

UNIVERSITY OF SHEFFIELD
EU contribution
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Address
WESTERN BANK
S10 2TN SHEFFIELD
United Kingdom

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