and their concentration was calculated based on amino acid analysis measurements. Solutions were aliquoted into sterile polypropylene vials, relyophilized and stored at 70 C until used. The molecular masses of the proteins (as determined by MALDI-MS) were consistent with the following sequences: NCp7 ¼ IQKGNFRNQRKTVKCFNCGKEG- HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQAN; NCp9 ¼ NCp7 + p1Gag ¼ NCp7 + FLGKIWPSHKGRP- GNF; NCp15 ¼ NCp7 + p1Gag + p6Gag ¼ NCp9 + LQSR- PEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLAS- LRSLFGSDPSSQ. Gag protein (lacking myristic acid at its N-terminus and the p6 domain at its C-terminus, referred to previously as Gag–Dp6) was puriﬁed from bacteria as described (2), except that EDTA was eliminated from the puriﬁcation procedure so that zinc ions would be retained in the zinc ﬁngers of the NC domain. This change required modiﬁcation of several steps in the procedure, as the protein with zinc bound bacterial nucleic acids considerably more tightly than that lacking zinc. Gag concentrations were measured from their absorbance in 6 M guanidine hydrochloride, pH 6.5. Fluorescence binding studies Poly(dT) was obtained in lyophilized form from Sigma (St Louis, MO). Poly(eA) was purchased from Pharmacia. LTR DNA oligonucleotides were purchased from Invitrogen with the following sequences: U5 sense, 50-TAA CTA GAG ATC CCT CAG ACC CTT TTA GTC AGT GTG GAA AAT CTC TAG CAG T-30; U5 antisense, 50-ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA TCT CTA GTT A-30; U3 sense, 50-ACT GGA AGG GCT AAT TCA CTC CCA AAG AAG ACA AGA TAT CCT TGA TCT GTG G-30; U3 antisense, 50-CCA CAG ATC AAG GAT ATC TTG TCT TCT TTG GGA GTG AAT TAG CCC TTC CAG T-30. Nucleic Acids Research, 2006, Vol. 34, No. 2 595 The oligonucleotides were aliquoted in ﬁve OD vials by the manufacturer and lyophilized. Once dissolved, the concen- tration of each solution was checked by UV absorbance. To anneal the oligonucleotides in solution, equimolar amounts of the complementary strands were mixed in Eppendorf tubes, heated to near boiling and allowed to cool slowly. A spectro- photometric temperature ramp showed >20% hyperchromism for the annealed duplex. Sodium monophosphate and biphos- phate, magnesium and sodium chloride were purchased from Aldrich Chemical Inc. (Milwaukee, WI). Fluorescence titrations were carried out with a SPEX Fluoromax-2 spectroﬂuorimeter. NC Trp emission was monitored at 350 nm (5 nm bandwidth) with 280 nm excitation (1 nm bandwidth) in a thermostated capped quartz cell (0.2 · 1.0 cm; Hellma Cells, Inc., Jamaica, NY); stepwise additions of nucleic acid to 0.5–1.0 mM NC solutions, in 10 mM sodium phosphate, pH 7.0 were performed. Readings were corrected for dilution and inner ﬁlter effects. NC protein– nucleic acid complex formation was monitored by reduction in the initial ﬂuorescence of the protein [or ethenoA ﬂuorescence enhancement, in the case of poly(eA)]. NC fractional saturation was inferred from the ratio of observed quenching to maximal quenching (at saturation), DFlim, for each of the nucleic acid additions. Limiting que- nching was calculated using the method of Kelly et al. (29). The plateau’s intersection with the initial slope indicates the occluded binding site size (n) for a titration carried out under near-stoichiometric conditions such as that shown in Figure 2A. Polynucleotide binding cooperativity was ana- lyzed using the model of McGhee and von Hippel (30). Salt-back titrations were carried out by addition of a 5.0 M NaCl solution to a preformed NC–nucleic acid complex (Figure 2B). Double-logarithmic plots of log K versus log [Na+] (Figure 2C) were used to derive binding constants under various ionic strength conditions (31). The binding free energy can thus be resolved into two components: an Figure 2. (A) Normalized fluorescence versus concentration ratio of nucleic acid to protein. Poly(dT) was added to NCp15 (0.5 mM in 1 mM sodium phosphate, pH 7.0, and 25 C). The plateau’s intersection with the initial slope indicates the stoichiometry (n, occluded binding site size in the case of a polynucleotide) for a titration carried out under near-stoichiometric conditions such as that shown. (B) Fluorescence recovery (F/F0) after the addition of 5 M NaCl aliquots to a pre-formed NCp15– poly(dT) complex. (C) Double-logarithmic plot for the association of NCp15 to poly(dT). The slope indicates the number of Na+ ions displaced; the y-intercept is log K0 at 1 M Na+, when only non-ionic interactions contribute to binding free energy.