Structural characterization of histone binding proteins

Vizjak, Petra (2015) Structural characterization of histone binding proteins. Diploma thesis, Faculty of Science > Department of Biology.

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Histones are the chief protein components of chromatin. Histone chaperone Nap1, together with karyopherin Kap114, shuttles H2A-H2B from cytosol to the nucleus. Nop1 methyltransferase in complex with Nop56 protein is responsible for H2AQ105 methylation, the first histone epigenetic mark dedicated to a specific RNA polymerase. The aim of this project was to structurally characterize histones in complex with these histone binding proteins. Standard molecular cloning techniques were used. Proteins were purified by affinity, ion exchange and size exclusion chromatography. Proteins were analyzed by SDS-poliacrylamide gel-electrophoresis and mass sprectrometry. We have shown that Nap1 (H. Sapiens) forms a complex with H2A-H2B (X. laevis) dimer. Nap1 terminal domains are not necessary for complex formation. Regions important for complex stability are protected from protease cleavage. Interaction between Nap1-H2A-H2B and Kap114 (S. Pombe) was confirmed. Complexes Nap1-H2A-H2B and Nap1-H2A-H2B-Kap114 were purified and precrystallization screenings were set up on crystallization robot. Nop1-Nop56 (K. lactis) complex was formed in vitro. Nop1 N-terminal domain is dispensable for interaction with Nop56. Complex forms soluble aggregates which has made purification difficult. Based on obtained results, new purification strategies will be developed. Further research will contribute to understanding of chromatin biology and the human diseases associated with histone chaperones.

Item Type: Thesis (Diploma thesis)
Keywords: fibrillarin, H2 import, H2A glutamine methylation, Kap114, Nap1, Nop56
Supervisor: Halić, Mario
Co-supervisor: Zoldoš, Vlatka
Date: 2015
Number of Pages: 59
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 18 Apr 2016 11:31
Last Modified: 18 Apr 2016 11:31

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