ALPHA-HELIX TO RANDOM COIL TRANSITIONS OF 2-CHAIN COILED COILS - EXPERIMENTS ON THE THERMAL-DENATURATION OF ISOLATED SEGMENTS OF ALPHA-ALPHA-TROPOMYOSIN

Circular dichroism (CD) experiments in the backbone (200-240 nm) region are reported for four isolated, excised two-chain, coiled-coil segments whose chains comprise, respectively, residues 11-127, 142-281, 1-189, and 190-284 of the rabbit alpha-alpha-tropomyosin (Tm) sequence. The uv and CD spectra for the noncross-linked segments are very similar to those for parent Tm. At 3-degrees-C, all have a helix content of 90% or more; moreover, all thermal denaturation curves depend on concentration, as required by mass action, and are completely reversible. At comparable concentrations, solutions show values of T1/2 (the temperature at which the helix content is 50%) following the order of 11Tm127 approximately 1Tm189 > 142Tm281 > 190Tm284. The thermal unfolding data for 11Tm127, 190Tm284, and 142Tm281 fall on apparently monophasic curves (single inflection point). However, curves for 1Tm189 show a heretofore unknown low temperature transition in which the helix content drops from approximately 90% at 2-degrees-C to approximately 73% at 20-degrees-C, indicating that this segment has one or more weak sections totaling approximately 50 residues per chain. Since thermal denaturation curves for noncross-lined 11Tm127, 142Tm281, and Tm have no such low temperature transition, i.e., the helix content is not additive, the weak region probably comprises the bulk of the residues between 127 and 189 in 1Tm189, but is somehow stabilized in 142Tm281 and in parent Tm. This nonadditivity may be explainable by a combination of loop entropy and local adjustments in packing of hydrophobes in the helix-helix interface. For C190-cross-linked 142Tm281, the uv and CD spectra and helix content are very similar to those for other coiled-coil species. In addition, the thermal unfolding curve shows a "pretransition" that is similar to but broader than that for cross-linked parent Tm and a "posttransition" that is almost identical to that for cross-linked parent Tm.