A novel protocol for the study of protein-nucleic acid interactions is presented and demonstrated
to be feasible. The protocol combines photochemical crosslinking techniques and mass
spectrometric methods into a new strategy for identifying protein domains or amino acid residues
that are in close contact with nucleic acid in protein-nucleic acid complexes. Identifying nucleic
acid binding domains in proteins provides a starting point for understanding structure-function
relationships in protein-nucleic acid complexes.
The protocol can be divided into three parts: 1) Cross linking of the protein-nucleic acid complex
by irradiation with ultraviolet light and subsequently verifying the crosslinking by mass
spectrometry; 2) Mass spectrometric peptide mapping of crosslinked protein-nucleic acid
complexes to identify crosslinked peptide-nucleic acid hybrids; 3) Tandem mass spectrometric
sequencing of peptide-nucleic acid hybrids to localize the crosslinked amino acid residue(s).
The experimental data described in this dissertation documents our efforts to establish and
implement this analytical protocol. Using several different protein-nucleic acid systems and
different crosslinking techniques, we have demonstrated the feasibility of a mass spectrometric
based approach to structurally characterize UV-crosslinked protein-nucleic acid complexes.
Matrix-assisted laser desorption/ionization mass spectrometry was for the first time demonstrated
to be highly effective for detection and molecular weight determination of intact, UV-crosslinked
protein-nucleic acid complexes and for molecular weight determination of synthetic and UV-crosslinked
peptide-nucleic acid hybrids. Electrospray ionization mass spectrometry and tandem
mass spectrometry was demonstrated to be effective for analysis of synthetic peptide-nucleic acid
hybrids and, in conjunction with HPLC, for peptide mapping of a protein. The first application of
MALDI mass spectrometry to the characterization of crosslinked peptide-nucleic acid hybrids
isolated from a photochemically crosslinked protein-nucleic acid complex demonstrate that the
new protocol can be used to identify nucleic acid binding domains in proteins. / Graduation date: 1995
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/35130 |
| Date | 20 October 1994 |
| Creators | Jensen, Ole Norregaard |
| Contributors | Barofsky, Douglas F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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