Abstract
Extensive studies have been conducted to elucidate the structure and function of the globular and C-terminal domains of linker histones. However, much is still unclear about the N-terminal domain (NTD). Determining the location of the N-terminal domain of linker histones in chromatin is necessary to resolve structural aspects of chromatin and elucidate its role. In this exploratory study, chemical crosslinking was employed to determine the proximity of the chicken H1.11L to other histones in chromatin. H1.11L was substituted with a single cysteine residue by site-directed mutagenesis. The cysteine substitution was made close to the N-terminus of the protein (H1.11L-S2C) and at the intersection of the basic and hydrophobic regions of the NTD (H1.11L-A21C). Chicken chromatin was reconstituted with mutant H1.11L molecules and the material cross-linked using hetero-bifunctional cross-linkers (Sulfo-SMCC, SPDP and Sulfo-MBS). Cross-linked samples were analyzed by SDS/PAGE and protein bands that represented cross-linked complexes were excised. Excised protein bands were in-gel digested with trypsin, GluC and propionylated prior to trypsin digestion and the resulting peptides analyzed by LC-MS/MS. Trypsin digestion resulted in the best sequence coverage and hence identification of the largest number of cross-links. While peptides originating from mutant H1.11L proteins as well as all four core histones were identified in most cross-linked complexes, a relatively small number of cross-links could be unambiguously assigned. The majority of cross-links were between mutant H1.11L molecules, confirming the close association of H1 in chromatin. Based on the location of the globular domain of H1 in chromatin, cross-links to the N-terminal domain of H3 and the C-terminal domain of H2A were expected. While these were not identified, a number of cross-links further away from the globular domain binding site were observed. This points to the extended nature of the N-terminal domain and the relatively large conformational space associated with it. The nucleosomal location of the identified cross-links is consistent with the notion that the basic region of the N-terminal domain traces the trajectory of nucleosomal DNA rather than interacting with linker DNA. However, more refined approaches are required to elucidate this further in future.
M.Sc. (Biochemistry)