Book of Abstracts: Albany 2005
The Tripeptide Recognizes AT Base Pairs of DNA
We have synthesized the fluorescent labeled tripeptide -- H-L-Thr-L-Val-L-Thr-N2H2-Dns (TVT) and its interaction with DNA was studied by circular (CD) and liner dichroism (LD), equilibrium dialysis, and fluorescence methods. It was shown: I. TVT molecules form dimers in solution. It follows from nonlinear dependence of fluorescence intensity on TVT concentration in the solution. II. TVT is adsorbed on DNA mainly as dimers. It follows from the S-like dependence of bound TVT concentration to DNA on TVT unbound concentration (by equilibrium dialysis). III. TVT dimers are localized in narrow groove of DNA upon binding. It follows from TVT competition with distamycine A for binding sites on DNA. IV. DNA-TVT complex may interact with each other by means of peptide-peptide interaction and form biduplex structures. It follows from nonlinear dependence of LD magnitude of TVT-DNA complex on DNA concentration in solution. V. TVT dimers are adsorbed to poly(dA) × poly(dT) better than to poly(dG) × poly(dC) polymers (see Fig. 1a,b). It is required (0.6-0.7) × 10-4 M of peptide to occupy a half binding sites on poly(dA) × poly(dT) and more than (2.5-2.8) × 10-4 M TVT to occupy a half binding sites on poly(dG) × poly(dC).
S. A. Streltsov1
1Engelhardt Institute of Molecular Biology
Figure 1: The dependence of fluorescence intensity (a) and CD magnitude (b) for complexes TVT-poly(dA) × poly(dT) -- (curve 1), and TVT-poly(dG) × poly(dC) -- (curve 2) on TVT concentration in solution. (a) Curve 1 (left axis) -- complex TVT with 7.84 × 10-5 M bp poly(dA) × poly(dT). Curve 2 (right axes) -- complex TVT with 4.27 × 10-5 M bp poly(dG) × poly(dC). The excitation wavelength = 380, the emission wavelength = 520 nm. Fluorescence intensity of unbound TVT was subtracted. (b) Curve 1 (left axis) -- complex TVT with 7.84 × 10-5 M bp poly(dA) × poly(dT), CD is measured at 270 nm. Curve 2 (rigth axis) -- complex TVT with 7.94 × 10-5 M bp poly(dG) × poly(dC), CD is measured at 255 nm. CD values of free DNA and unbound TVT were subtracted. The cell optical path length 4 mm. Buffer: 1 mM sodium cacodylate, pH 6.8, 25% (v/v) MeOH.
The work was partly supported by the Program of the Basic Studies of the Russian Academy of Sciences Presidium, N10. Molecular and Cell Biology.