Studies of the folding mechanisms of monomeric proteins have been a major focus in efforts to describe the protein folding code: how a protein’s primary structure encodes its unique, three-dimensional structure as well as the pathway to this native state from an unfolded, random coil ensemble of states. The folding kinetics of small, single-domain model systems can often be described by a rapid, two-state mechanism (for reviews, see Jackson 1998; Plaxco et al. 2000; Daggett and Fersht 2003). In contrast, the folding kinetics of larger, multidomain monomeric proteins are often more complex, requiring the coordination of long-range interdomain and short-range intradomain interactions (for reviews, see Kim and Baldwin 1990; Matthews 1993; Wallace and Matthews 2002). Significant insights have been gained from monomeric folding systems regarding the intramolecular forces responsible for the productive formation and stabilization of secondary and tertiary structures as the folding energy landscape is successfully and efficiently traversed. However, it is not yet clear how these insights apply to the intermolecular association reactions that stabilize the quaternary structure of oligomeric proteins. Oligomeric proteins are prevalent in biology and offer evolutionary advantages such as the potential of increased thermostability relative to mono mers and sensitive regulation through allostery and cooper ativity in ligand binding. The folding of oligomeric proteins requires that the protein folding code direct the formation of intramolecular secondary and tertiary structure as well as the productive quaternary interactions necessary for biologi cal function. Folding experiments on large, oligomeric en zymes demonstrated the complexity of this coordination of multiple levels of structure formation (for reviews, see Jaenicke 1987; Jaenicke and Lilie 2000). A lack of reversibil ity, often resulting from aggregation, is a complication in the folding of many proteins, and particularly oligomeric systems. Protein concentration-dependent, nonproductive aggregation reactions often kinetically compete with on pathway, partially folded, kinetic intermediates in large oligomeric proteins (Seckler 2000). In vivo, this competition between productive folding and aggregation is often offset by the assistance of chaperones. Given these complexities, recent efforts have focused on smaller oligomeric folding model systems. Small (less than 60 residues per monomer), dimeric, two-state folding sys tems have been identified, for example the P22 Arc repres sor (Milla and Sauer 1994; Srivastava and Sauer 2000) and the GCN-4-derived leucine zipper peptides (Zitzewitz et al 1995, 2000). However, just as with monomeric systems, larger dimers, containing subdomains, tend to traverse more complicated folding landscapes, with transient kinetic inter mediates and sometimes parallel pathways. Examples in clude ketosteroid isomerases (Kim et al. 2001a,b), Esche richia coli Trp repressor (Gittelman and Matthews 1990; Mann et al. 1995; Gloss et al. 2001), glutathione transfer ases (Wallace et al. 1998; Wallace and Dirr 1999), and bacterial luciferase (Clark et al. 1997; Noland et al. 1999; Inlow and Baldwin 2002). Both monomeric and dimeric kinetic intermediates have been observed, but as yet, no clear rules are discernable for the prediction of the features of the folding landscape of dimeric proteins. This article investigates the folding reactions of the his tone fold motif, using the (H3–H4)2 tetramer (Fig. 1 ▶) as model system. The histone fold is a dimerization motif found in several important protein–DNA complexes. The fold was first structurally characterized in the eukaryotic heterodimers of the core nucleosome—the (H3–H4)2 tetramer and the H2A–H2B heterodimer (Arents et al. 1991; Luger et al. 1997a). The motif is also seen in the struc tures of homodimeric archael histones (Starich et al. 1996; Zhu et al. 1998; Decanniere et al. 2000) and some of the TATA-binding protein Associated Factors (TAFs) of the TFIID complex (Xie et al. 1996; Birck et al. 1998). Sequence alignments have suggested that many other DNA binding proteins may also contain the histone fold dimerization motif (Arents and Moudrianakis 1995; Baxevanis et al. 1995). Figure 1. Ribbon structure of the histone fold and the dimer–dimer interface of the H3–H4 tetramer. The H3 monomers, from residues 38–135 of 135, are shown in a lighter gray; the darker-colored H4 monomers encompass residues 20–102 ... The histone fold contains a long central α-helix that is flanked on both the N and C termini by short β-loops and shorter α-helices (Arents et al. 1991; Luger et al. 1997a). The histone monomers associate to form dimers in a head-to-tail fashion called the “handshake motif” (Arents and Moudrianakis 1995). Two H3–H4 dimers then dimerize with one another through a four-helix bundle of their H3 C-terminal helical regions. Thus, the folding of the (H3–H4)2 tetramer requires the coordinated folding of four polypeptide chains and the assembly of two dimerization interfaces. To date, biophysical characterization of the eukaryotic histones has focused on the equilibrium stabilities of the oligomers (Baxevanis et al. 1991; Karantza et al. 1995, 1996 Karantza et al. 2001; Gloss and Placek 2002; Placek and Gloss 2002; Banks and Gloss 2003). The equilibrium stability of recombinant (H3–H4)2 tetramer was determined by GdnHCl-induced denaturation, using fluorescence and circular dichroism spectroscopies (Banks and Gloss 2003). At moderate ionic strength (μ ~ 0.2 M), H3–H4 is unstable, with little native baseline in the unfolding transition. Therefore, tri-methylamine-N-oxide (TMAO) was used to stabilize the tetramer to obtain accurate thermodynamic parameters. The equilibrium unfolding of the (H3–H4)2 tetramer was best described by a three-state mechanism, with well-folded H3–H4 dimers as a populated intermediate. When compared to the structurally similar H2A–H2B dimer, the H3–H3′ tetramer interface and the H3–H4 histone fold are strikingly less stable (Banks and Gloss 2003). This article provides the first report of kinetic folding studies and a proposed folding mechanism for an oligomer containing the histone fold.