Structural studies on the transmembrane domain of Neu and the ?-trefoil protein hisactophilin
A point mutation (Val664 [right arrow] Glu) within the transmembrane (TM) region of Neu, a receptor tyrosine kinase (RTK), promotes dimerization leading to enhanced catalytic activity and cellular transformation. Because of the technical challenges associated with the characterization of membrane-associated proteins, the elucidation of the underlying mechanism behind the dimer-promoting ability of the transforming mutation represents a significant challenge. In Chapters 1 through 4, the effect of the polar mutation is explored through a study of chemically synthesized peptides, incorporating the complete TM domain of both proto-oncogenic and mutant Neu, by nuclear magnetic resonance (NMR) spectroscopy, in both membrane-mimetic solution conditions and in lipid bilayers. In the solvent trifluoroethanol (TFE), both peptides exist as monomers, exhibit very similar chemical shift profiles, and adopt [alpha]-helical structures possessing a distinct bend one helical turn downstream of the mutation site. The data in this solvent are inconsistent with a structural change in the TM domain of Neu upon mutation. In micellar dispersions, both peptides adopt similar levels of helicity and helix-helix association. However, the chemical shift profiles, nuclear Overhauser effect (NOE) connectivities, and amide exchange rates for the two peptides exhibit much more variability than in the solvent TFE, and these differences are most pronounced close to and upstream of the transforming mutation site. 1H-NMR spectra were acquired for the two peptides dispersed in dimyristoylphosphatidylcholine (DMPC) vesicles under magic angle spinning (MAS) conditions. Unfortunately, broad line-widths and poor signal-to-noise levels prevented the acquisition of well resolved two-dimensional heteronuclear spectra needed for the assignment of resonances, and a qualitative comparison of the structures adopted by the two peptides. In Chapter 5, hydrogen/deuterium exchange rates are presented for the slowly exchanging amide protons in the [beta]-trefoil protein hisactophilin. Analysis of the rates as a function of pH, denaturant concentration, and temperature provide insight into the dynamic properties of the protein at equilibrium. Most amides in this protein exchange via the EX2 mechanism under stabilizing conditions. Hisactophilin does not adopt partially structured equilibrium intermediates, possibly because most of the [beta]-strands contribute residues to the large hydrophobic core of the protein that is cooperatively disrupted upon unfolding.