600 Nucleic Acids Research, 2006, Vol. 34, No. 2 been previously discussed. In Discussion, we present com- ments on the general information that can be obtained from studying the hysteresis of single DNA molecule stretching, followed by several conclusions regarding the proteins studied in this work. We will show that, although the information from studying the non-equilibrium features of the DNA stretch– relax cycle is not quantitative, it can serve as an important basis for understanding the kinetics of protein–nucleic acid interactions. Fluorimetric titrations with HIV-1 NC proteins The nucleic acid binding properties of HIV-1 NC in its three differently processed forms were investigated with the long single-stranded homopolymeric DNA and RNA molecules, poly(dT) and poly(eA), respectively (45). Both polynuc- leotides were bound by the proteins nearly stoichiometrically under the conditions of the reverse titration experiment (see Materials and Methods). Binding to poly(dT) resulted in a large reduction (86%) in the ﬂuorescence emission of the sole Trp residue (Trp37) in NCp7, as reported previously (45,46). The presence of the additional Trp residue (Trp61) in NCp9 and NCp15 resulted in a decrease in the limiting quenching at saturation for these NC sequences (Table 2), in agreement with earlier reports that Trp61 is not directly involved in the interaction of NCp9 with nucleic acids (46). Indeed, the binding afﬁnity and thermodynamic parameters derived from salt-back titrations were nearly identical for NCp7 and NCp9. The occluded binding site size increased from NCp7 to NCp9 to NCp15, reﬂecting the larger size of the binding protein. NCp15 bound poly(dT) with an afﬁnity an order of magnitude larger than NCp7 or NCp9. However, this effect was not observed for poly(eA) titrations. The binding free energy in the standard state (1 M NaCl) was comparable for all three proteins, indicating that the binding afﬁnity of Table 2. Nucleic acid binding properties of HIV-1 NC proteinsa System NCp7 + poly(dT) NCp9 + poly(dT) NCp15 + poly(dT) NCp7 + poly(eA) NCp9 + poly(eA) NCp15 + poly(eA) NCp7 + 30LTR-dsDNA NCp9 + 30LTR-dsDNA NCp7 + 50LTR-dsDNA NCp9 + 50LTR-dsDNA NCp7 + 30LTR/U5 NCp9 + 30LTR/U5 NCp7 + 50LTR/U3 NCp9 + 50LTR/U3 nav 6.4 9.5 10.3 6.6 10.2 11.8 2.7 bp 4.3 bp 2.9 bp 4.4 bp 6.6 8.3 6.8 9.5 DFlim (%) 86 83 70 +135 +210 +195 85 61 86 59 92 81 93 86 K (M 1) 9.3 · 107 1.1 · 108 1.4 · 109 1.4 · 109 3.8 · 109 1.7 · 109 8.3 · 108 3.8 · 109 3.4 · 108 8.9 · 109 4.3 · 107b 6.2 · 107b 2.2 · 108b 3.9 · 108b dlog K/ dlog [Na+] log K0 2.54 2.61 3.35 2.02 2.06 2.04 3.26 3.65 3.21 3.96 2.31 2.30 2.96 2.85 2.89 2.83 2.45 5.54 5.11 5.40 2.40 2.28 2.49 2.03 3.30 3.46 2.77 3.23 aThermodynamic results from fluorimetric binding isotherms of NCp7, NCp9 and NCp15 with various polynucleotides were obtained by two methods: (i) addition of nucleic acid aliquots to protein (Trp quenching) and (ii) addition of protein aliquots to poly(eA), both in a solution of 10 mM sodium phosphate buffer, pH 7.0, at 25 C. Average errors were as follows: ±1 for nav; ±1.6% for DFlim; ±28% for K; ±0.07 for log(K)/log([Na+]); and ±0.19 for log (K0). K0 is the association constant extrapolated to 1 M salt. bThese results were measured only at higher salt than that reported for the other measurements in the table. To allow for comparison of results, they have been extrapolated to 10 mM sodium phosphate buffer conditions. NCp15 would tend to converge with that of NCp7 and NCp9 at the higher ionic strength typical of physiological conditions. dsDNA binding was also examined with long terminal repeat (LTR) sequences (Table 2). An increase in occluded binding site size was again seen in the association of NCp9 with duplex DNA, compared with that of NCp7. Owing to the scarcity of the material, NCp15 binding titrations were not carried out. Somewhat lower limiting quenching levels were obtained for NCp7 and NCp9, as compared with the related single-stranded sequences, in agreement with results reported for NCp7 using other duplex DNA molecules (47). Trp quenching was again highest for the single-Trp NCp7, pointing to a lack of involvement of the second Trp residue in NCp9 or NCp15, in agreement with their similar non- electrostatic contribution (log K0) to the binding free energy, regardless of the presence of the C-terminal segment contain- ing Trp61. Binding of all proteins to double-stranded LTRs had a larger electrostatic component than to ssDNA and was characterized by a smaller site size. NCp9 bound almost an order of magnitude tighter than NCp7 to LTR sequences. Overall, the bulk ﬂuorescence titration results conﬁrm the ﬁndings from the stretching experiments, which is that the equilibrium binding afﬁnities of NCp7, NCp9 and NCp15 to nucleic acids are of similar magnitude. Fluorescence anisotropy binding studies with Gag and NCp7 Because the DNA stretching studies were done with DNA only, we wanted to test whether or not our conclusions con- cerning Gag and Gag cleavage products containing NC were applicable to Gag and NC binding to RNA. The interactions of NC with either RNA or DNA are both potentially biolo- gically important in the context of NC-induced facilitation of reverse transcription. To this end, we performed FA binding studies of Gag and NCp7 binding to 10 nt DNA and RNA oligonucleotides, d(TG)5 and r(UG)5. The results of these measurements are presented in Table 3. The measured values of the binding constant for NC are in agreement with its value obtained in our stretching and tryptophan ﬂuorescence quenching studies (see Tables 1 and 2). It seems likely that this agreement is in fact a result of mutual compensation of two effects. The FA binding studies were performed at higher (150 mM NaCl) salt, as compared with 50 mM NaCl in DNA stretching and 10 mM NaCl in tryptophan quenching experiments. Higher salt decreases the binding afﬁnity of NC to nucleic acids. However, this weakening is compensated by the fact that TG and UG oligonucleotide sequences bind NC more strongly than random nucleic acid sequences (48,49). Perhaps the most important observation is that both NC and Gag bind similarly to RNA and DNA Table 3. Binding constants of HIV-1 NCp7 and Gag proteins to 10 nt ssRNA and ssDNA oligomers measured by FA Oligonucleotide NCp7 Gag K (M 1) (UG)5 (8.1 ± 1.2) · 107 (5.2 ± 2.2) · 107 K (M 1) (TG)5 (7.2 ± 1.5) · 107 (3.4 ± 0.9) · 107 Binding measurements were performed as described in Materials and Methods.