Folding and stability of a primitive protein

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (Delta G < 1 kcal mol(-1)) which convert rapidly (approximate to 6 x 10(4) M-1 s(-1)) into dimers (Delta G = 9.8 kcal/mol) and then more slowly (approximate to 3 M-1 s(-1)) into tetramers (Delta G = 28 kcal mol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution. (c) 2005 Elsevier Ltd. All rights reserved.