Recognition between disordered polypeptide chains from cleavage of an alpha/beta domain: self-versus...

Advances in structural biology have provoked a re-evaluation of the biological significance of the disordered state of proteins. We believe that the rules that govern structure, stability and kinetics in the molecular recognition between disordered polypeptide chains can be elucidated by studying processes that couple association with folding. The reassembly of single domain proteins by fragment complementation provides an excellent opportunity to study them. Since almost the complete sequence is available, although not on a single chain, most of the complementary fragments are expected to reassemble. However, that happens not to be the case. We have chosen E. coli thioredoxin (Trx), a small, single alpha/beta-domain protein, as a model system to study the effect of the site and number of cleavages on the reassembly of complementary fragments. We have shown at atomic detail the reassembly after cleavage of a loop (1-73, 74-108) and after cleavage of an alpha-helix (1-37, 38-108). Although both sets of fragments produce native-like complexes, there are clear differences in the interface geometry, apparent stability of the folded state and mechanism of association/folding: (i) the apparent equilibrium dissociation constant for 1-37/38-108 complex (4 microM) is higher than the one for 1-73/74-108 complex (49 nM), (ii) the apparent rate constants of non-self-association are similar (about 10(3) M-1s-1), and (iii) only the 1-37 fragment self-associates under these experimental conditions. Here the competition between self- and non-self-association leads to an apparently less stable 1-37/38-108 complex.