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Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA

(RECEIVED September 5, 2007; FINAL REVISION November 6, 2007; ACCEPTED November 7, 2007)

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central -helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with G°(H₂O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.

Keywords: protein folding; circular dichroism; fluorescence; kinetic intermediates; equilibrium stability

Reprint requests to: Lisa M. Gloss, School of Molecular Biosciences, Washington State University, Box 644660, Pullman, WA 99164-4660, USA; e-mail: lmgloss{at}wsu.edu; fax; (509) 335-9688.

Abbreviations: BP, burst-phase, occurring in the stopped-flow 5-ms dead time; CD, circular dichroism; G° (H₂O), the free energy of unfolding in the absence of denaturant; F_app, apparent fraction of unfolded monomer; FL, intrinsic Tyr or Trp fluorescence; GdmCl, guanidinium chloride; hMfB, histone B from the archae Methanothermus fervidus; hPyA1, histone A1 from the archae Pyrococcus strain GB-3a; KPi, potassium phosphate, pH 7.2; m value, parameter describing the sensitivity of the unfolding transition to the [GdmCl]; MRE, mean residue ellipticity, normalization of CD data for protein concentration and number of residues; N₂, native dimer; NCP, nucleosome core particle; SF, stopped-flow; 2M, two partially folded dissociated monomers; 2U, two unfolded, disassociated monomers.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073224308.

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