Dissertation
STABILIZING INTERACTIONS IN THE H2A-H2B MONOMERIC AND DIMERIC KINETIC INTERMEDIATES
Doctor of Philosophy (PhD), Washington State University
01/2011
Handle:
https://hdl.handle.net/2376/3488
Abstract
The long-standing goal of protein folding studies is to understand how the amino acid sequence encodes the folding of a peptide from an extended conformation to the biologically active, three-dimensional structure. Theoretically, evolution should have smoothed the folding landscapes of proteins to reduce any intermediates or deviation from the most direct and rapid trajectory of folding, i.e. kinetic traps. However, transiently populated intermediates are common features in the folding landscapes of large proteins. Previous comparison of homologous dimeric histone proteins, H2A-H2B, hMfB, hPyA1, and hFoB, identified that transient population of intermediates correlated with rapid association, suggesting intermediates are not kinetic traps, but may accelerate folding. Understanding the structural features of kinetic intermediates will help elucidate their role in the folding landscape.
The folding mechanism of H2A-H2B is multi-state, with transient population of a dimeric ensemble (I2 and I2*) within the 5 ms stopped-flow mixing dead-time. The I2 ensemble folds to the native dimer, N2, by an observable first-order reaction. To determine the regions of structure in the I2 ensemble, 26 Ala mutants of buried hydrophobic residues were characterized, spanning all three helices of the canonical histone fold as well as the C-terminal helix of H2B. All but one targeted residue contributed significantly to the stability of I2, the transition state and N2; however, only mutations in the hydrophobic core of the dimer interface perturbed the population of I2*.
The isolated H2A and H2B monomers are partially folded at equilibrium and kinetically competent to fold to the I2 ensemble and N2. Therefore, the partially folded monomers are a good model of a monomeric kinetic intermediate likely populated prior to dimerization to form the I2 ensemble. The same 26 Ala mutations were studied in the monomers to determine if the collapsed structure in the monomers was conserved in the I2 ensemble, a likely source of the non-native structure observed in I2*. Of the 14 destabilizing monomer mutants, 12 destabilized I2*, suggesting the monomer structure is conserved in I2*. A putative association mechanism of H2A-H2B was developed from the mutational studies of the kinetic intermediates and is discussed.
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Details
- Title
- STABILIZING INTERACTIONS IN THE H2A-H2B MONOMERIC AND DIMERIC KINETIC INTERMEDIATES
- Creators
- Paul J. Guyett
- Contributors
- Lisa M. Gloss (Advisor)William B. Davis (Committee Member)John H. Nilson (Committee Member)Luying Xun (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 149
- Identifiers
- 99900581751601842
- Language
- English
- Resource Type
- Dissertation