Structural Studies of a Non-Sequence-Specific DNA Binding Protein, Hmg-D, Complexed With DNA
Dow, Linda Kay
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https://hdl.handle.net/2142/85489
Description
Title
Structural Studies of a Non-Sequence-Specific DNA Binding Protein, Hmg-D, Complexed With DNA
Author(s)
Dow, Linda Kay
Issue Date
1999
Doctoral Committee Chair(s)
Mair E.A.Churchill
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
An HMG box is a DNA binding domain common to a large number of proteins whose diversity in function range from transcriptional activation and repression to chromatin assembly. These proteins can be viewed as two classes divided by their ability to bind DNA in a sequence-specific vs. non-specific manner: LEF-1 and SRY fall into the former category while the latter is exemplified by HMG-1/2 and by HMG-D, a non-histone chromosomal protein from Drosophila melanogaster. Regardless of specificity, these proteins adopt a conserved 'L' shaped, three helix DNA binding motif as seen by NMR. While NMR structures of the sequence-specific proteins complexed with DNA have been determined, structures of the non-specific proteins bound to DNA have been hindered by the flexibility at the protein/DNA interface and by the overall size of the resulting complex. A complex of HMG-D with DNA has been designed for use in NMR studies that addresses these issues by recruiting the protein to a single site on the DNA using a cross-linked DNA sample and protein that is isotopically enriched to 100% with 13C and 15N and is 60% randomly deuterated. Using this sample for NMR studies has confirmed that HMG-D maintains its global fold upon complexation with DNA. Protein/DNA contacts can be traced to the regions of the protein adjacent to a putative DNA intercalation site at Met13, and to the N terminus of helix two. The third helix is not disturbed by complexation. The basic tail is unstructured in solution, contrary to previous predictions, and crucial DNA contacts confirm the importance of HMG-D's basic tail. The design of the complex, the strategies used for both the chemical shift assignments and structure determinations, the resulting protein structure, and the observations of the protein/DNA contacts are presented. The similarities and differences between the complex and free protein are also discussed.
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