NMR studies to elucidate the binding of a mutantlac repressor headpiece to operators and the structure of the signal peptide of rat aldehyde dehydrogenase in various environments

Christine A. Jensen Karslake, Purdue University

Abstract

The binding of both the wild-type and the Y7I mutant lac repressor headpieces to various tetradecamer symmetric operators was studied by phosphorus NMR spectroscopy. The binding of these headpieces was extremely similar in the phosphorus atoms contacted and the magnitude of their titration. Specific binding was found to occur in mutant operators where the chemical shifts of the phosphorus atom, which is a manifestation of the geometry of the phosphate, were not greatly altered. However, when the chemical shifts of important phosphates were altered by a mutation, the specific binding no longer took place. The proton assignments for the Y7I mutant headpiece were completed. This headpiece contains three $\alpha$-helical regions which are analogous to those found in the wild-type headpiece. The I7 displays very similar interactions to those of Y7 observed in the wild-type headpiece. However, the first two helices which have been previously shown to contact DNA were found to be weaker for this mutant compared to the wild-type. Approximately 170 short-, medium- and long-distance NOEs were assigned to constrain the molecule for structural determination. The Y7I mutant headpiece was complexed with the tetradecamer symmetric operator and intermolecular NOEs were noted. These intermolecular NOEs designate where headpiece binds to its operator. Again, isoleucine 7 displayed binding to guanosine 11-H8 as tyrosine 7 did in the wild-type headpiece. These data suggest that the Y7I and the wild-type headpiece bind to the symmetric operator in a similar fashion. The structure of a twenty-two amino acid peptide derived from rat aldehyde-hydrogenase's signal peptide was studied by proton NMR spectroscopy in dodecyl-phosphocholine micelles. This peptide contains two amphiphilic $\alpha$-helical regions one at the C-terminus and the other at the N-terminus. The C-terminal helix was determined to be the stronger of the two as determined by NH-NH cross-peak intensity and exchange experiments. The NH of leucine 18 and leucine 19 were exchanged very slowly in the micellar environment. The structure of this same signal peptide was determined in a 2,2,2-trifluoro-ethanol solution. The structure was shown to be similar to that structure in dodecylphosphocoline micelles except that the NH of leucine 18 and leucine 19 did not exchange nearly as slowly as in the micelle.

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry

COinS