CORRELATION BETWEEN CONFORMATIONAL AND BINDING-PROPERTIES OF NEBULIN REPEATS

Nebulin, a large protein (600 to 800 kDa) located in the thin filament of striated vertebrate muscle, is assumed to bind and stabilise F-act in. Complete sequence determination of human nebulin has only recently been accomplished showing a uniform modular structure along the whole length of the molecule. Up to 97% of the sequence is assembled from r epeats of a sequence motif 35 amino acid residues long. This architect ure suggests that a structural and functional understanding of such a large molecule may be possible by characterising single repeats and re constructing from them the behaviour of the whole molecule. In the pre sent study, we extend and generalise to the whole molecule previous wo rk carried out on single repeats from a limited region of nebulin. Kno wledge of the complete sequence allowed extensive analysis of the sing le repeats revealing a progressive N to C-terminal divergence that is mirrored by an increase of the alpha-helix propensity. A number of syn thetic peptides spanning the sequences of selected repeats were obtain ed and their conformational and binding properties studied in detail. All the peptides showed a tendency to fold as transient helices in aqu eous solution with helix content as observed by CD and NMR studies in excellent agreement with predictions. A higher helical tendency of rep eats near the C terminus was observed. Analysis of the influence of ch arged media as well as trifluoroethanol on the folding of single repea ts strongly suggested that the mechanism by which the nebulin alpha-he lix is stabilised is mostly electrostatic. Peptides with higher helica l content also showed a higher binding affinity to F-actin. Considerab ly varying effects were observed for the peptides on F-actin viscosity and polymerisation. We discuss the divergence in sequence and helical tendency and its correlation to the functional data with regard to th eir significance for the assembly of the thin filament during myogenes is. (C) 1996 Academic Press Limited