The Application of a Reporter Group to α-Chymotrypsin

<p>A reporter group method was developed in order to obtain information about small changes in the environment at specific positions in protein molecules. In this method one environmentally sensitive group is introduced into a specific position in the protein so that small changes induced by substrates or modifiers can be followed. In this study the synthesis of a potential reporter group reagent, 2-bromoacetamido-4-nitrophenol, was accomplished. This reagent has a moiety which is sensitive to the polarity of the environment and to pH, and which reports changes in its environment to an appropriate detector. It contains a second moiety which reacts with methionine residues. This potential reporter group was attached to chymotrypsin by a covalent bond to a methionine residue. It was demonstrated that substrates perturb the spectrum of the reporter-modified chymotrypsin and that the spectral perturbations have characteristics related to a tentative but consistent interpretation of changes in the absorption spectrum of the reporter group in terms of the environment of the group. The applications of reporter groups to the study of conformational changes of proteins and to the study of the three-dimensional orientations of substrates at the active sites of the proteins are discussed.</p> A reporter group method was developed in order to obtain informa-tion about small changes in the environment at specific positions in protein molecules. In this method one environmentally sensitive group is introduced into a specific position in the protein so that small changes induced by substrates or modifiers can be followed. In this study the synthesis of a potential reporter group reagent, 2-bromoacetamido-4-nitrophenol, was accomplished. This reagent has a moiety which is sensitive to the polarity of the environment and to pH, and which reports changes in its environment to an appropriate detector. It contains a second moiety which reacts with methionine residues. This potential reporter group was attached to chymotrypsin by a covalent bond to a methio-nine residue. It was demonstrated that substrates perturb the spectrum of the reporter-modified chymotrypsin and that the spectral perturbations have characteristics related to a tentative but consistent interpretation of changes in the absorption spectrum of the reporter group in terms of the environment of the group. The applications of reporter groups to the study of conformational changes of proteins and to the study of the three-dimensional orientations of substrates at the active sites of the proteins are discussed.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

1965

Author dc:creator

• Hille, Merrill Burr

Contributors dc:contributor

• Daniel Koshland Jr.

• reporter group method
• environmentally sensitive moiety
• 2-bromoacetamido-4-nitrophenol
• chymotrypsin modification
• spectral perturbations
• protein conformational changes
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/572

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1576