Two-dimensional NMR structural studies of peptides in micelle and 40 percent TFE
Abstract
We have determined the structure of a mutant of rat liver aldehyde dehydrogenase (ALDH) in a dodecylphosphocholine micelle and in 40% trifluoroethanol. To test the role of the linker region of ALDH signal sequence in the import and processing of the precursor protein, we deleted it from the ALDH signal peptide and precursor protein. The 19 amino acid signal peptide of ALDH, to which has been added 3 residues at the C-terminus and from which has been deleted the 3 residue flexible linker region, has been studied by two dimensional NMR in a dodecylphosphocholine micelle and in 40% trifluoroethanol (TFE). In both of these environments the peptide is shown to contain a single $\alpha$-helical segment that extends almost the entire length of the peptide. NH exchange experiments show residues on the hydrophobic face of the peptide to exchange much more slowly than those of the hydrophilic face in the micelle. NH exchange experiments in both micelle and 40% TFE suggest that the C-terminal portion of the helix is significantly more stable than the N-terminal segment. Combined with previous studies, these results suggest that precursor protein import simply requires a sufficiently long amphiphilic helix (or helices) to stably bind to the membrane. The linker deleted ALDH peptide contains a single helix of 12-14 residues that is long enough to provide a hydrophobic surface that can stably interact with the hydrophobic interior of the membrane. We have also used $\sp1$H NMR to determine the structure of glucagon-like peptide (7-36 amidated) in a dodecylphosphocholine micelle. This peptide hormone is shown to have a structure similar to that observed for glucagon. It consists of an N-terminal random coil segment (residues 1-7), two helical segments, (8-14) and (18-29) and a linker region (15-17). The C-terminal helix is shown to be more stable than the N-helix as determined by amide proton exchange experiments. $\sp{19}$F NMR has been used to study two trifluoromethylketone inhibitors of the serine proteases subtilisin BPN and $\alpha$-chymotrypsin. Both inhibitors are shown to bind in two different conformations as observed by $\sp{19}$F NMR.
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Biochemistry
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