IMINO PROTON-EXCHANGE PROVIDES AN H-1-NMR FOOTPRINT OF PROTEIN-DNA INTERACTIONS - GENERAL STRATEGY AND APPLICATION TO THE SRY HMG BOX

A novel H-1 nuclear magnetic resonance (NMR) strategy for ''footprinti ng'' specific protein-DNA target sites is demonstrated Relative rates of site-specific imino-proton exchange in the free and bound DNA duple x are determined by use of laminar-shifted shaped pulses in NOESY spec tra 2D exchange crosspeaks between imino (omega(2) dimension) and wate r (omega(1) dimension) resonances in principle provide site-specific p robes of protein binding. Chemical exchange is distinguished from nucl ear Overhauser enhancements (NOEs) to bound water by use of POESY spec troscopy. This strategy is illustrated in H-1-NMR studies of the SRY h igh-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, p reviously identified by random binding-site selection as an optimal SR Y target sequence. The extent of damping correlates with sites of prot ein-DNA contacts in the minor groove but not with the magnitude of 1H- NMR complexation shifts. SRY binding has recently been shown to introd uce significant distortions in DNA structure. The DNA is sharply bent and underwound; the minor groove is widened and major groove compresse d. 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.