Structural features of phi29 single-stranded DNA-binding protein. I. Environment of tyrosines in terms of complex formation with DNA.
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi29 is absolutely required for viral DNA replication in vivo. About approximately 95% of the intrinsic tyrosine fluorescence of phi29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi29 SSB fluorescence emission (phiF = 0.067 +/- 0.010), (ii) the fast motions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega (cooperativity) defining phi29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = K omega, being largely independent of temperature. Altogether, the fluorescent properties of phi29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.
Soengas
M S
MS
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid, Spain.
Mateo
C R
CR
Salas
M
M
Acuña
A U
AU
Gutierrez
C
C
D017103:Bacillus Phages / Q000737:chemistry*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D012995:Solubility; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*; D014779:Virus Replication
D004268:DNA-Binding Proteins; D012997:Solvents; D014764:Viral Proteins; D014443:Tyrosine