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

Objectif

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 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 SYNTHESISE 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 MAIN GOAL OF THE PROJECT IS TO ACHIEVE A BETTER UNDERSTANDING OF THE FACTORS REQUIRED FOR THE HIGH RECOVERY OF HUMAN FERRITINS FROM E. COLI THE SPECIFIC AMINO ACID SEQUENCES THAT DETERMINE THE CORRECT FOLDING, ASSEMBLY, THERMOSTABILITY AND IMMUNOLOGICAL PROPERTIES OF FERRITIN AND TO EXPLORE THE POSSIBILITY TO USE THIS HIGHLY STABLE AND REPETITIVE PROTEIC STRUCTURE AS SCAFFOLD FOR SPECIFIC BINDING SITES. THE FIRST YEAR OF THE PROJECT WILL BE MOSTLY DEDICATED TO THE PRODUCTION AND CHARACTERIZATION OF THE STRUCTURES OF THE RECOMBINANT H AND L FERRITINS, IN COMPARISON WITH THE NATURAL ONES. THE SECOND AND THIRD YEAR WILL BE DEDICATED TO CRYSTAL GROWTH AND X-RAY DATA COLLECTION, IN COLLABORATION WITH THE GROUP IN THE UNIVERSITY OF SHEFFIELD (UK), LOCALISATION OF EPITOPES ON THE MOLECULES AND THE CONTINUATION OF MUTAGENESIS EXPERIMENTS DIRECTED AT SPECIFIC SITES OF THE ALPHAHELICES.

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Appel à propositions

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Régime de financement

CSC - Cost-sharing contracts

Coordinateur

THE UNIVERSITY OF MILANO
Contribution de l’UE
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Adresse
Via Cicognara 7
20133 MILANO
Italie

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