Folding and Stability of a Primitive Protein

• Published in: Journal of molecular biology Vol. 348; no. 5; pp. 1261 - 1272
• Main Authors: Riechmann, Lutz, Lavenir, Isabelle, de Bono, Stephanie, Winter, Greg
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 20.05.2005
• Subjects: Amino Acid Sequence; combinatorial protein; Dimerization; Directed Molecular Evolution; domain swapping; Evolution, Molecular; Kinetics; Molecular Sequence Data; non-homologous recombination; protein evolution; Protein Folding; Protein Structure, Tertiary; Proteins - chemistry; Proteins - genetics; segment swapping; Thermodynamics; combinatorial protein; protein evolution; domain swapping; segment swapping; SELDI; non-homologous recombination
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.03.029

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 (Δ G<1 kcal mol −1) which convert rapidly (≈6×10 4 M −1 s −1) into dimers (Δ G=9.8 kcal/mol) and then more slowly (≈3 M −1 s −1) into tetramers (Δ 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.