The primary objective of this project is to understand the structure-function relationship of a peptide which produces a voltage switchable ion channel in a phospholipid membrane. We are using three different carboxy-terminal fragments from the colicin E1 protein. One was obtained with CNBr cleavage and has only 152 amino acids. The other two are resistant cores obtained after thermolysin and trypsin digestion which contain about 185 residues. All of these peptides have been shown to have the same ion channel activity and switching properties as the intact purified colicin protein prior to cleavage. There are three problems of general importance which will be addressed in these studies: 1) What is the three-dimensional structure of the peptide when it forms the ion channel. 2) How does the binding of the protein to membranes take place and, 3) What is the mechanism by which an ion channel can be switched between and OPEN and a CLOSED state by changing the voltage across the membrane. Our plan is to use site-specific mutagenesis to shorten the fragment at both ends until we determine the minimum length necessary for ion channel activity. We will insert charges into the uncharged region and alter other charges in order to determine which residues are important for the binding of the protein to the membrane and which are facing into the lumen of the channel. Biochemical and enzymatic studies will be carried out with the C-terminal fragments and intact proteins made by wild type colicin E1 genes and by various mutants. All of these will be analyzed in terms of the conductance they produce in phospholipid membranes: their biochemical properties; their ability to bind to lipid and their biological activities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031540-02
Application #
3279612
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-12-01
Project End
1986-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Graduate Schools
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027