Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein

Tropomyosin is an alpha -helical coiled-coil protein that aligns head-to-ta il along tl e length of the actin filament and regulates its function. The solution structure of the functionally important N terminus of a short 247- residue non-muscle tropomyosin was determined in an engineered chimeric pro tein, GlyTM1bZip, consisting of the first 19 residues of rat short a-tropom yosin and the last 18 residues of the GCN4 leucine zipper. A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli. Tripl e resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances. Multidimensional nuclear Overhauser effect spectr a, X-filtered spectra and (3)J(H-N-H-alpha) scalar coupling were analyzed u sing AutoStructure. This is the first application of this new program to de termine the three-dimensional structure, of a symmetric homodimer and a str ucture not previously reported. Residues 7-35 in GlyTM1bZip form a coiled c oil, but neither end is helical. Heteronuclear N-15-H-1 nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. T he C-13' chemical shifts of the coiled-coil backbone carbonyl groups in Gly TM1bZip showed a previously unreported periodicity, where resonances arisin g from residues at the coiled-coil interface in a and d positions of the he ptad repeat were displaced relatively upfield and those arising from residu es in c positions were displaced relatively downfield. Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha -helical backbone and side-chains a t the coiled-coil interface broadened or shifted with T-M values similar to 20 degreesC lower than the loss of alpha -helix measured by circular dichr oism, suggesting the presence of a folding intermediate. The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple con formations. The conformational flexibility of the N termini of short tropom yosins may be important for their binding specificity. (C) 2001 Academic Pr ess.