Very Fast Folding and Association of a Trimerization Domain from Bacteriophage T4 Fibritin Sarah Gu ¨ the 1 †, Larisa Kapinos 1 †, Andreas Mo ¨ glich 1 † Sebastian Meier 2 , Stephan Grzesiek 2 and Thomas Kiefhaber 1 * 1 Division of Biophysical Chemistry, Biozentrum der Universita ¨t Basel Klingelbergstrasse 70, CH-4056 Basel, Switzerland 2 Division of Structural Biology Biozentrum der Universita ¨t Basel, Klingelbergstrasse 70 CH-4056 Basel, Switzerland The foldon domain constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4. Its function is to promote folding and trimerization of fibritin. We investigated structure, stability and folding mechanism of the isolated foldon domain. The domain folds into the same trimeric b-propeller structure as in fibritin and undergoes a two-state unfolding transition from folded trimer to unfolded monomers. The folding kinetics involve several consecutive reactions. Structure formation in the region of the single b-hairpin of each monomer occurs on the submillisecond timescale. This reaction is followed by two consecutive association steps with rate constants of 1.9(^ 0.5) £ 10 6 M 21 s 21 and 5.4(^ 0.3) £ 10 6 M 21 s 21 at 0.58 M GdmCl, respectively. This is similar to the fastest reported bimolecular association reactions for folding of dimeric proteins. At low concentrations of protein, folding shows apparent third-order kinetics. At high concentrations of protein, the reaction becomes almost independent of protein concen- trations with a half-time of about 3 ms, indicating that a first-order folding step from a partially folded trimer to the native protein (k ¼ 210ð^20Þ s 21 ) becomes rate-limiting. Our results suggest that all steps on the folding/ trimerization pathway of the foldon domain are evolutionarily optimized for rapid and specific initiation of trimer formation during fibritin assembly. The results further show that b-hairpins allow efficient and rapid protein–protein interactions during folding. q 2004 Elsevier Ltd. All rights reserved. Keywords: protein folding; protein association; trimeric proteins; prolyl isomerization; fast folding *Corresponding author Introduction Fibritin is a rod-like structural protein of bacteriophage T4, which is attached to the neck of the virion via its N-terminal domain to form the collar structures (“whiskers”). Fibritin consists of an N-terminal anchor domain (residues 1–46), a large central coiled-coil part (residues 47–456) and a small C-terminal globular domain (residues 457 – 486). 1 The 30 amino acid residue C-terminal domain was termed foldon, since it was shown to be essential for fibritin trimerization and folding in vivo and in vitro. 1–3 Each subunit of the foldon domain consists of a single b-hairpin, which assemble into a b-propeller-like structure in the trimer. 1 The trimer is stabilized by hydro- phobic interactions involving Trp476 of each subunit, intermolecular salt-bridges between Glu461 and Arg471, and intermolecular backbone hydrogen bonds between Tyr469 and Arg471 (Figure 1). Expression of the isolated foldon domain (residues 457–483) yields a stable trimer, which shows a cooperative two- state thermal unfolding transition. 4 Residues 484–486 were omitted from this study, since this region is unordered in the X-ray structure of fibritin. 1 The foldon domain was proposed to be an evolutionarily optimized trimerization/folding 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. † S.Gu ¨ ., L.K. and A.M. contributed equally to this work. E-mail address of the corresponding author: t.kiefhaber@unibas.ch Abbreviations used: F-moc, N-(9-fluorenyl)- methoxycarbonyl; GdmCl, guanidinium chloride; RDC, residual dipolar coupling. doi:10.1016/j.jmb.2004.02.020 J. Mol. Biol. (2004) 337, 905–915