STRUCTURE-FUNCTION STUDIES OF A SWITCHABLE ION CHANNEL

The ion channel region of the colicin El protein will be studied as a model for voltage switchable ion channels found in all organisms. Site-directed mutagenesis with chemically synthesized oligonucleotides have been used to introduce methionine codons in the gene in order to provide new cyanogen bromide cleavage sites and produce short C-terminal fragments which can be tested for their channel forming activity. At present, a C-terminal fragment of only 88 amino acids has been found to be active in producing channels in a phospholipid membrane while one with only 75 amino acids was not active. Additional methionine codons will be introduced to establish the critical minimum length of the active peptide. We also have determined stochiometrically that only on peptide molecule is enough to make the channel and that in the experimental conditions used, this molecule is a monomer. The one cysteine residue in the wild type molecule has been changed to glycine and nine new cysteine codons introduced, one at a time. Many more of these amino acid changes will be made in the part involved in the channel formation and the proteins produced will be studied by attaching specific chemical groups to the SH groups located throughout the peptide chains. The effects of these sulfhydryl reagents will be examined by either electron- spin resonance spectroscopy if the attached molecule's have an electron spin or they will be studied by determining the alterations in th conductance of single channels when the attached molecules are big since the channel will then be blocked if a large group is pointing inward. Using the information obtained from these modified proteins, one can determine which residues in the peptide point into the lumen and which point out into the lipid. In this way, one can determine which parts of th protein are in the form of a beta-sheet where the residues would point alternatively in or out with a repeat period of 2 and which are in the form of an alpha-helix in which case the repeat period would be 3.6 residues. These experimental measurements will be used to help in deducing the structure of the channel. New channels could be produced by altering the amino acids facing the lumen. In all cases, extensive computations will be done in order to make theoretical estimates of the channel conductance and their changes and these will be compared with the observed values.