Imino proton exchange provides an 1H-NMR footprint of protein-DNA interactions: general strategy and application to the SRY HMG box.
A novel 1H nuclear magnetic resonance (NMR) strategy for "footprinting" specific protein-DNA target sites is demonstrated. Relative rates of site-specifc imino-proton exchange in the free and bound DNA duplex are determined by use of laminar-shifted shaped pulses in NOESY spectra. 2D exchange crosspeaks between imino (omega2 dimension) (omega1 dimension) resonances in principle provide site-specific probes of protein binding. Chemical exchange is distinguished from nuclear Overhauser enhancements(NOEs) to bound water by use of ROESY spectroscopy. This strategy is illustrated in 1H-NMR studies of the SRY high-mobility group (HMG) box, the Y-chromosome-encoded "master switch" for testis determination in man. In a specific complex between the protein and a 15-basepair DNA site, imino-proton exchange was observed to be damped selectively within the six basepair subsite 5'-ATTGTT, previously identified by random binding-site selection as an optimal SRY target sequence. The extent of damping correlates with sites of protein-DNA contacts in the minor groove but not with the magnitude of 1H-NMR complexation shifts. SRY binding has recently been shown to introduce significant distortions in DNA structure. The DNA is sharply bent and underwound; the minor groove is widened and major groove compressed. Our results demonstrate that despite such distortions base pairing is stably maintained. Protein binding in the DNA minor groove shields DNA imino protons from exchange with solvent.
Weiss
M A
MA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
King
C Y
CY
D001482:Base Composition; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006609:High Mobility Group Proteins / Q000737:chemistry*; D006801:Humans; D007097:Imines / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011522:Protons
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D007097:Imines; D011522:Protons