Abstract
Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
In order to substantiate this proposal and obtain quantitative determinations of Kobs and W, we carried out a series of binding measurements with the homopolymer poly(dT) for recombinant Gene 32 protein (g32P)-B, purified from the 0.5 M NaCl elution pool of two separate runs of the ssDNA-cellulose column,5 and the g32P-(A + B) core protein, derived from g32P-B by proteolysis and re-purified via ssDNA-cellulose as described above
Represent the theoretical curve obtained when those parameters derived from an analysis of the data cast in the linear format of Fig. 9C are recast, indicating an acceptable fit to Cooperative binding by Gene 32 protein (g32P) to ss nucleic acids requires the N-terminal B domain as evidenced by quantitative equilibrium binding experiments carried out with the tryptic core g32P-(A + B) fragment on the polynucleotide, poly(dT), i.e. w = 1 or noncooperative binding [11]
We describe overexpression, purification, and characterization of the N-terminal tryptic deletion fragment of T4 gene 32 protein, g32PZ2-301or g32P-B
Summary
In order to substantiate this proposal and obtain quantitative determinations of Kobs and W, we carried out a series of binding measurements with the homopolymer poly(dT) for recombinant g32P-B, purified from the 0.5 M NaCl elution pool of two separate runs of the ssDNA-cellulose column,5 and the g32P-(A + B) core protein, derived from g32P-B by proteolysis and re-purified via ssDNA-cellulose as described above.
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