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STRUCTURE-FUNCTION RELATIONSHIPS IN A PEPTIDE HORMONE AND ENZYMES. THE APPLICATION OF PROTEIN ENGINEERING

Objective

THE LONG RANGE GOAL IS TO OBTAIN THE KNOWLEDGE NECESSARY FOR INTRODUCING APPROPRIATE CHANGES IN ENZYMES OF INDUSTRIAL INTEREST.
How changes in a protein's structure modify its functions. The approach was based on research into insulin and the enzymes amylase and lipase, the goal being to determine the structure of the proteins and the modified proteins and the modified proteins by X-ray crystallography. With this technique, the position of the modified groups in insulin and the enzymes and their interactions with the surrounding proteins can be accurately determined and related to changes in function. This detailed structural information is essential for designing new changes in function of insulin and the enzymes. New technologies were also developed for increasing the quality of the data from X-ray experiments.

The 3-dimensional structures of mutated insulins were determined and the role the changed amino acid side chains had in the modified properties of the hormone was assessed. Insulin's properties of self assembly are best affected by introducing new charges which repel each other when the individual molecules come together. The structures of 2 amylases were determined, revealing details of their catalytic sites. Calcium ions were developed to bind at the catalytic groups and thus abolish enzymatic activity. One of the 2 enzymes is active in acid conditions. The 2 structures are now being compared. Finally, the crystal structure of a fungal lipase was also solved. This showed that the catalytic group in this enzyme is very similar to the catalytic group in the serine proteases.
THE RESEARCH WILL CONCENTRATE ON THE X-RAY ANALYSIS OF AN ENZYME OF INDUSTRIAL INTEREST.
CRYSTALS WILL BE GROWN TO A SUITABLE SIZE FOR X-RAY ANALYSIS. THE CRYSTALS WILL BE CHARACTERIZED, AND DATA COLLECTED AT HIGH RESOLUTION (2.5 A) AND PROCESSED.
CRYSTALS WILL BE PREPARED WITH ISOMORPHOUS HEAVY ATOM SUBSTITUTIONS. THE HEAVY ATOM DERIVATIVES OF THE PROTEIN CRYSTAL WILL BE ANALYSED. THE ELECTRON DENSITY OF THE PROTEIN IN THE CRYSTAL WILL BE CALCULATED. THE ATOMIC PARAMETERS WILL BE REFINED,. FINALLY STRUCTURAL ANALYSIS AND MODELLING WILL BE CARRIED OUT.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

University of York
Address
Heslington -
YO1 5DD York
United Kingdom

Participants (1)

NOVO INDUSTRY A/S
Denmark
Address

Bagsvaerd