Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence

The regulatory properties of naturally occurring tropomyosins (Tms) of diff ering lengths have been examined. These Tms span from 4 to 7 actin subunits . Native proteins have been used to study the common 7 actin-spanning skele tal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively). The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 [Maytum, R., et al. (2000) Biochemistry 39, 11913]. The regul ation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive SI binding titration. The equilibrium between closed and open (C to M states, K-T = 0.1-0.14) was similar for all vertebrate Tms. A part from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14). The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5). The determined value of K-T depended on the N-terminal sequence (K-T = 0.15-1) . These results are compared with the effect of S1 upon Tm's affinity for a ctin. The yeast Tms have regulatory parameters similar to those of skTm, bu t unlike skTm, S1 has little effect upon their actin affinity. This shows t hat an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vert ebrate Tms is probably the result of a direct interaction of S1 with Tm.