The successful synthesis of globin in E.coli and its reconstitution with haem to fully functional haemoglobin has opened up a wide field of research. We plan to use a combination of directed mutagenesis, X-ray crystallography and computer graphics to follow evolution backwards from mammals to birds, reptiles, fish and invertebrates. We wish to understand the structural basis for the species-specific properties of haemoglobins, and to test my hypothesis that new functions arise from the replacement of a few amino acid residues in key positions, while the majority of amino acid differences between species are functionally neutral or near neutral and are due to genetic drift. Other possibilities are the synthesis of a stable tetrameric haemoglobin; induction of changes in the electron distribution of the haem by amino acid substitutions in the haem pocket, and study of their physiological effects; the substitution of amino acid residues in the haem pocket to generate catalytic activities; study of the transmission of elecytrostatic effects through the protein by measuring the change in pKa's of specific histidines as a function of carboxylates or lysines placed at different distances from them. We plan further development of antisickling drugs. I should like to solve the structure of human serum albumin by X-ray analysis in order to study the stereo-chemistry of drug binding by this protein. I should also like to solve the structure of horseradish peroxidase in order to understand its catalytic mechanism and, by directed mutagenesis, improve its utility as an analytical tool in clinical biochemistry. We will use the system of eukaryotic protein synthesis in E.coli for making other proteins of medical or biological interest. I shall continue the X-ray analysis of abnormal hemoglobins whose physiological properties need to be understood on a stereochemical basis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031461-05
Application #
3342590
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1983-12-01
Project End
1989-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Medical Research Council Lab of Molec Biol
Department
Type
DUNS #
232560263
City
Cambridge
State
Country
United Kingdom
Zip Code
CB2 2QH
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