Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence.
The regulatory properties of naturally occurring tropomyosins (Tms) of differing 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 skeletal 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 regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration. The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms. Apart 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 KT depended on the N-terminal sequence (KT = 0.15-1). These results are compared with the effect of S1 upon Tm's affinity for actin. The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity. This shows that 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 vertebrate Tms is probably the result of a direct interaction of S1 with Tm.
Maytum
R
R
Department of Biosciences, University of Kent at Canterbury, Canterbury, UK. rmm@ukc.ac.uk
Konrad
M
M
Lehrer
S S
SS
Geeves
M A
MA
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis; D046911:Macromolecular Substances; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014335:Tropomyosin