Kinetics of self-assembly of alpha alpha-tropomyosin coiled coils from unfolded chains.

Abstract

The two-chain coiled-coil structural motif is found in fibrous muscle proteins and leucine zippers. Unfolding/refolding studies abound, but none establishes the time scale or mechanism of structural assembly from separated, unfolded chains. Stopped-flow circular dichroism studies of such refolding of a-tropomyosin chains are reported here. The two-chain coiled coil, in which two right-handed a-helices align in parallel and in register with left-handed intertwining, is found in disparate biological contexts. Proteins from muscle and bacterial cell walls as well as certain DNA-binding proteins (leucine zippers) share this architecture. Amphipathicity, resulting from a pseudo-repeating heptad of amino acids in which residues one and four are hydrophobes and five and seven are oppositely charged, provides the energetic impetus for such dimeric association and probably for an important fraction of the total stability as well. A recent review of these and many other features of coiled coils is available (3).Many physical studies of thermally or denaturant-induced unfolding establish the equilibrium helix content of particular coiled coils (4-11). Nevertheless, important questions remain concerning the contribution of intra-vs. interchain interactions, hydrophobic vs. electrostatic forces, and the number and nature of populated conformational states at equilibrium (1, 7). Moreover, the time scale and mechanism by which separate, unfolded chains self-assemble into the registered, parallel double helix are totally unknown.We address here the latter problem. We report studies in which denatured chains ofrabbit a-tropomyosin are suddenly brought into a benign medium in a stopped-flow apparatus and the time course of coiled-coil generation is measured by circular dichroism (CD). The data are interpreted to provide a physical picture of the mechanism of the process and the structure of the intermediate. MATERIALS AND METHODSPreparation of aa-tropomyosin from cardiac muscle of genetically selected New Zealand White rabbits was as previously described in detail (9). Each a chain in the dimeric molecule has 284 residues, residue 190 being cysteine. Reduced and disulfide-crosslinked forms were produced by dithiothreitol (DTT) reduction and ferricyanide oxidation, respectively, in the standard manner (9-11).Reduced tropomyosin was unfolded in U6",oNaCl5oo-NaPi50DTTj(7.4) (where U signifies urea and Suc signifies sucrose). The unfolded tropomyosin was diluted in the stopped-flow mixer with 9 volumes of Suc450NaCl500NaPi50-(7.4). Thus, refolding took place in U60DSuc405NaCl500NaPi50-DTTo.1(7.4). Sucrose assures that the diluent and the unfolding medium match in viscosity and density, thus avoiding mixing anomalies.CD measurements were made with a Jasco 500A instrument and SFC-5 stopped-flow attachment, using an 80-ms flow time. The dead time was 40 ms, as estimated by calibration with the reaction of Ni2+ with D-tartrate (12). The data ofellipticity (at 222 nm) vs. time were computer-fit to the desired kinetic equation usin...

Molecular basis of coiled-coil formation

“…Interacting helices then ''zip up'' along the molecule to form the stable coiled-coil structure. Notably, this hierarchic folding mechanism (18,45,46) also provides a rational explanation for the efficient assembly of extensive coiled-coil domains (47) where chains are unlikely to align in-register if coiled-coil interactions form randomly along the sequence.…”

Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif

“…In the unfolding direction, only one exponential model is required to fit the kinetic transients, such that the barrier between U and I 2 is Equation 13.…”

“…Of these proteins, the parallel dimeric coiled coil appears to be one of the best understood (11)(12)(13)(14)(15)(16), as this protein contains only ␣-helical elements of secondary structure. Indeed, a wealth of reports have been published on the folding of the yeast transcription factor GCN4-p1 (12,14,(17)(18)(19)(20)(21) with many reporting the protein to fold in a two-state mechanism.…”

ββ homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation–renaturation

“…Experiments on tropomyosins from various sources have been interpreted to support each of the three mechanisms, thus raising the possibility that the mechanism is system dependent Brown & Schachat, 1985;Lehrer et al, 1989;Lehrer & Qian, 1990;Mo et al, 1991;Ozeki et al, 1991). Experimental data on the fundamental processes involved are needed to allow choice among the mechanisms for a given system.…”