596 Nucleic Acids Research, 2006, Vol. 34, No. 2 electrostatic contribution calculated from the slope of the regression line in the double-logarithmic plot, and a hydro- phobic (or non-electrostatic) contribution from the y-intercept (32). In the absence of net anion uptake or release during binding, dividing the slope by the number of ions bound (in the thermodynamic sense) per phosphate group in the double-stranded or single-stranded nucleic acid lattice, yields the number of electrostatic interactions incurred by the pro- tein. The y-intercept represents the logarithm of the binding afﬁnity at 1 M Na+ (log K0), the standard state where electrostatic interactions are considered null. Low salt buffer was used in the initial titrations to maximize nucleic acid binding to NC proteins, permitting also a broader range of salt concentrations to be explored in the subsequent salt-back titration. Fluorescence anisotropy binding studies FA is based on the observation that when a ﬂuorescent molecule is excited with plane-polarized light, it emits polar- ized ﬂuorescent light into a ﬁxed plane if the molecules are stationary between excitation and emission (33,34). The FA is determined by measuring the ratio of the emission intensities from the parallel and perpendicular planes. Because the FA of a molecule is proportional to its molecular volume, this ratio is used to study molecular interactions. d(TG)5 labeled with ﬂuorescein at the 30 end was synthes- ized by Leo Lee, SAIC-Frederick and puriﬁed by HPLC. (UG)5 labeled with ﬂuorescein at the 30 end was from Dharmacon (Chicago, IL). To determine the dissociation con- stants of Gag and NCp7 to these oligonucleotides, 10 nM stock solutions of either d(TG)5 or (UG)5 were prepared in HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.5) with 100 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 5 mM b-mercaptoethanol and 1 mM ZnCl2 and aliquoted into 96-well Costar polypropylene plates (Corning, NY). NC and Gag proteins were serially diluted into both oligonucleotides. An aliquot of 40 ml was removed and transferred into 384-well Costar polypropylene plates (Corning, NY). Samples were excited at 485 nm and the emission intensities at 535 nm from the parallel and perpendicular planes were measured using a Tecan Ultra plate reader (Durham, NC). Reactions were performed as three independent experiments. Curves were ﬁt assuming a single binding site per oligonucleotide, using the relation (34): AnðCÞ ¼ ððR·Anbound AnfreeÞ·ðfðKd þCþLtÞ½ð Kd þCþLtÞ2 4·Lt·C 1=2g=2LtÞþAnfreeÞ ððR 1Þ·ðfKd þCþLtÞ½ð Kd þCþLtÞ2 4Lt·C 1=2g=ð2LtÞÞþ1Þ where An(C) is the measured anisotropy as a function of con- centration, Lt is the oligonucleotide concentration, Anfree is the anisotropy of unbound oligonucleotide, Anbound is the aniso- tropy of the completely bound oligonucleotide solution, R is the ratio of the ﬂuorescence intensity of saturated bound oligonucleotide relative to free oligonucleotide, C is the protein concentration and Kd is the measured dissociation constant resulting from the ﬁt. Because we do not actually know that there is only a single binding site per oligonuc- leotide, below we refer to the Kd measurements resulting from this ﬁt as ‘apparent Kd’. 1 Force-induced melting The optical tweezers instrument and data acquisition were described previously (35,36). Biotinylated DNA was obtained as previously described (37). The DNA was labeled at the 30 ends only such that it would be free to rotate when stretched. All proteins were aliquoted in volumes of 10 ml and 10 or 25 mM protein concentration in speciﬁc NC-storage buffer (20 mM HEPES, 5 mM b-mercaptoethanol, 0.1 mM TCEP, pH 7.5), and stored at 80 C before use. NC proteins were reconstituted with two molar equivalents of zinc. The tethering buffer used in this study for all force-induced melting experiments (FIM buffer) contained 10 mM HEPES with 45 mM NaCl and 5 mM NaOH, pH 7.5. Lambda DNA, dNTPs (dATP, dTTP, dGTP), enzymes and buffers were purchased from Promega, the biotin-11-dCTP from Enzo, HEPES, NaCl and NaOH from Sigma, and the streptavidin- coated beads from Bang Laboratories. After capturing a single DNA molecule in the tethering buffer, the molecule was stretched to verify that the correct force-extension curve was obtained in the absence of bound protein. To measure the effect of the proteins on this transition, a buffer solution containing a ﬁxed concentration of protein dissolved in FIM buffer was added to the experimental cell until the buffer surrounding the captured DNA molecule was completely exchanged. RESULTS Below we will present our results in two main sections. In the ﬁrst, we use a single DNA molecule stretching technique and in the second we use a tryptophan ﬂuorescence assay. These experiments are designed to elucidate the speciﬁc features of the interaction with DNA of the HIV-1 NC protein while in the context of Gag as well as in its three differently processed forms. The overall goal is to understand how the sequential proteolysis of HIV-1 NC affects its nucleic acid interaction properties, and to determine whether this can be correlated with the change of NC’s function in various stages of the viral replication cycle. In a third section, we compare the binding of NC and Gag with a very short oligoribonucleotide and a very short oligodeoxynucleotide to determine whether the 20 OH (or the methyl group in thymidine) plays a signiﬁcant role in the interactions of these proteins with nucleic acids. Single DNA molecule stretching studies of NC–DNA and Gag–DNA interactions In this work we apply a single DNA molecule stretching technique to obtain information on DNA interactions with each of the HIV-1 NC and Gag proteins. Some information about the NCp7–DNA interaction obtained by this method was already discussed in our previous work (35,36). In this report we perform a much broader analysis of the HIV-1 NC– DNA interaction as a function of protein concentration, which allows us to determine the binding constant of each protein to DNA. In addition, we analyze the equilibrium and non- equilibrium features of our stretching curves, which allow us to determine both equilibrium and kinetic properties of protein–DNA interactions.