Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate.
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 alpha-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 DeltaG degrees (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.
Stump
Matthew R
MR
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
Gloss
Lisa M
LM
D019843:Archaeal Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D019281:Dimerization; D006657:Histones / Q000737:chemistry*; D007700:Kinetics; D017012:Methanobacteriales / Q000737:chemistry; D050505:Mutant Proteins / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019711:Pyrococcus / Q000737:chemistry; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D019843:Archaeal Proteins; C090526:HPyA1 protein, Pyrococcus; D006657:Histones; D050505:Mutant Proteins; C094495:histone HMf; D014443:Tyrosine; D014364:Tryptophan