CONFORMATION OF THE CIRCULAR DUMBBELL D(PCGC-TT-GCG-TT) - STRUCTURE DETERMINATION AND MOLECULAR-DYNAMICS

The circular DNA decamer 5'-d < pCGC-TT-GCG-TT >-3' was studied in sol ution by means of NMR spectroscopy and molecular dynamics in H2O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (den oted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Cr ick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 con former becomes increasingly dominant (95% at 314 K). This conformer ha s a partially disrupted G(anti)-C(syn) closing base pair in the 5'-GTT C-3' loop with only one remaining (solvent-accessible) hydrogen bond b etween NHalpha of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5'-CTTG-3' loop remains stable. The two conformers occur in s low equilibrium (rate constant 2-20 s(-1)). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxati on matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. coupling constant a nalysis ((3)J(HH), (3)J(HP), (3)J(CP)) gave detailed information about the local geometry around backbone torsion angles beta, gamma, delta, and epsilon, revealing a relatively high flexibility of the 5'-GTTC-3 ' loop. The values of the coupling constants are virtually temperature -independent. 'Weakly constrained' molecular dynamics in solvent was u sed to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over < r(-3)> to predic t dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S-2 factor s and < r(-6)> averaging of the < r(-3)>-averaged relaxation matrices. On matrix averaging: the agreement of NOE volumes with experiment imp roved significantly for protons located in the thermodynamically less stable 5'-GTTC-3' loop. The difference in stability of the 5'-CTTG-3' and 5'-GTTC-3' loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacki ng overlap of the base pairs closing the minihairpin loops. The syn co nformation for dC(1), favored at high temperature, is stabilized by so lvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle chi.