The overall goal of this proposal is to characterize the structure of histone kinetic folding intermediates and address the unresolved issue of whether such species are traps that hinder productive folding OR are means to accelerate folding, and if so, how. This proposal will expand understanding of protein folding to dimers, where formation of secondary and tertiary structure must be coordinated with the appropriate association of polypeptides, while avoiding inappropriate association leading to aggregation and fibrillation. As a folding model, histones have three key features: 1) as the protein core of the nucleosome, their characterization will elucidate nucleosome function;2) they exemplify protein sequence degeneracy?high structureconservation with low sequence similarity. Degeneracy is a key stumbling block to prediction methods, that is best approached by studying homologous structures;and 3) despite a conserved fold, the eukaryotic H2A-H2B heterodimer and the archael hMfB and hPyA1 homodimers fold by different kinetic mechanisms, permitting study of how monomeric and dimeric intermediates contribute to rapid association and folding.
Two specific aims will test the following hypotheses: 1) Faster folding is achieved by population of kinetic intermediates;destabilizing the intermediates will slow folding. The structure of monomeric and dimeric kinetic intermediates will be determined, using CD, FL, Cys protection and mutagenesis to modulate their stabilities to determine if their population favors rapid folding. 2) Kinetic intermediates which accelerate folding are favorable, even in macromolecularly crowded solutions that promote off-pathway oligomerization of partially folded species. The folding efficiency of the three histones, which fold with and without intermediates, will be examined in solutions crowded with high concentrations of inert polymers. The long term goals of the studies are two-fold: to define general rules, including the impact of intermediates, on how poorly folded polypeptides efficiently recognize other macromolecules while avoiding inappropriate associations, such as with self, that lead to pathological oligomers;and to develop a detailed molecular, thermodynamic and kinetic description of nucleosome assembly and dynamics. The results of this proposal and the long term goals have two aspects of medical relevance. First, many human diseases involve protein misfolding, including amyloid fibrils. These pathological structures typically arise from intermediates. Domain-swapped oligomers, like the histone fold, seem particularly susceptible to pathological oligomerization. Second, stability and transiently populated species of histones dictate nucleosome dynamics and function. Nucleosomal packaging of DMA regulates processes such as transcription, replication and repair. When these processes go awry because of misregulation of nucleosome function, assembly and dynamics, disease states result, particularly cancer.
|Chen, Yujie; Tokuda, Joshua M; Topping, Traci et al. (2017) Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core. Proc Natl Acad Sci U S A 114:334-339|
|Chen, Yujie; Tokuda, Joshua M; Topping, Traci et al. (2014) Revealing transient structures of nucleosomes as DNA unwinds. Nucleic Acids Res 42:8767-76|
|Guyett, Paul J; Gloss, Lisa M (2012) The H2A-H2B dimeric kinetic intermediate is stabilized by widespread hydrophobic burial with few fully native interactions. J Mol Biol 415:600-14|
|Topping, Traci B; Gloss, Lisa M (2011) The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones. Protein Sci 20:2060-73|
|Stump, Matthew R; Gloss, Lisa M (2010) Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer. J Mol Biol 401:518-31|
|Gloss, Lisa M (2009) Equilibrium and kinetic approaches for studying oligomeric protein folding. Methods Enzymol 466:325-57|
|Stump, Matthew R; Gloss, Lisa M (2008) Mutational analysis of the stability of the H2A and H2B histone monomers. J Mol Biol 384:1369-83|
|Stump, Matthew R; Gloss, Lisa M (2008) Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate. Protein Sci 17:322-32|
|Hoch, Duane A; Stratton, Jessica J; Gloss, Lisa M (2007) Protein-protein Forster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z. J Mol Biol 371:971-88|