STRUCTURAL FEATURES OF PHI-29 SINGLE-STRANDED DNA-BINDING PROTEIN .1.ENVIRONMENT OF TYROSINES IN TERMS OF COMPLEX-FORMATION WITH DNA

The single-stranded DNA-binding protein (SSB) of Bacillus subtilis pha ge phi 29 is absolutely required for viral DNA replication in vivo. Ab out similar to 95% of the intrinsic tyrosine fluorescence of phi 29 SS B is quenched upon binding to ssDNA, making tyrosine residues strong c andidates to be directly involved in complex formation with ssDNA. Thu s, we have studied the spectroscopic properties of the phi 29 SSB tyro sines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolve d fluorescence measurements. phi 29 SSB tyrosines do not seem to be hi ghly restricted by strong interactions with neighbor residues, as sugg ested by (i) the high value of the average quantum yield of the phi 29 SSB fluorescence emission (Phi(F) = 0.067 +/- 0.010), (ii) the fast m otions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), an d (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer an alysis of the quenching by acrylamide and I- indicates that phi 29 SSB tyrosines are surrounded by a negatively charged environment and loca ted in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDN A binding allowed us to determine that temperature has an opposite eff ect on the thermodynamic parameters Il (intrinsic binding constant) an d omega (cooperativity) defining phi 29 SSB-poly(dT) interaction, the effective DNA binding constant, K-eff = K omega, being largely indepen dent of temperature. Altogether, the fluorescent properties of phi 29 SSB tyrosines are consistent with a direct participation in complex fo rmation with ssDNA.