N-TERMINI OF ECORI RESTRICTION-ENDONUCLEASE DIMER ARE IN CLOSE PROXIMITY ON THE PROTEIN SURFACE

The N-terminal region of EcoRI endonuclease is essential for cleavage yet is invisible in the 2.5 Angstrom crystal structure of endonuclease -DNA complex [Kim, Y., Grable, J. C., Love, R., Greene, P. J., Rosenbe rg, J. M. (1990) Science 249, 1307-1309]. We used site-directed fluore scence spectroscopy and chemical cross-linking to locate the N-termina l region and assess its flexibility in the absence and presence of DNA substrate. The second amino acid in each subunit of the homodimer was replaced with cysteine and labeled with pyrene or reacted with bifunc tional cross-linkers. The broad absorption spectra and characteristic excimer emission bands of pyrene-labeled muteins indicated stacking of the two pyrene rings in the homodimer. Proximity of N-terminal cystei nes was confirmed by disulfide bond formation and chemical cross-linki ng. The dynamics of the N-terminal region were determined from time-re solved emission anisotropy measurements. The anisotropy decay had two components: a fast component with rotational correlation time of 0.3-3 ns representing probe internal motions and a slow component with 50-1 00 ns correlation time representing overall tumbling of the protein co njugate. We conclude that the N-termini are close together at the dime r interface with limited flexibility. Binding of Mg2+ cofactor or DNA substrate did not affect the location or flexibility of the N-terminal region as sensed by pyrene fluorescence and cross-linking, indicating that substrate binding is not accompanied by folding or unfolding of the N-terminus.