He Supplemental Information. Zeff is a measure on the variety of Na+ ions displaced from RNA upon protein binding and reflects the amount of the positively charged groups at the protein surface straight interacting using the RNA phosphates (Record et al. 1976; Rouzina and Bloomfield 1997). In remedy, 70 and 90 of your phosphate backbone is occupied by Na+ ions for ssDNA and dsDNA, respectively (Record et al. 1976). Since the Na+ occupancy with the structured RNAs studied right here (Fig. 1) probably falls among these two numbers, our reported Zeff values have not been normalized to account for partial occupancy. We ensured that the Zeff and Kd(1M) values calculated were insensitive to the number of protein-bind-Webb et al.Consistent with the notion of weak GagRNA binding cooperativity was also the Psi observation that Psi RNA binds to Gag, a b c d CANC, and NC with related Zeff values Na1/2 (mM) Kd(1M) (M) Z eff Gagp6 Variant A and with Kd(1M) values, differing only by WT 0.15 0.006 372 three (five.2 1) 10-5 5.0 0.two around twofold for Gag and NC. WT (750 nM) 0.16 0.008 439 7 (6.3 3) 10-5 5.6 0.5 Zeff reflects all ions displaced upon nucleic -4 CANC 0.094 0.001 188 four (two.five 1) ten 4.0 0.3 -5 acid binding–ions displaced by direct NC 0.087 0.003 272 6 (two.6 0.eight) 10 3.7α-Hydroxy-4-cholesten-3-one Protocol 3 0.Biliverdin Metabolic Enzyme/Protease 2 contacts involving the RNA and protein 4.9 0.three WM 0.12 0.003 305 five (1.0 0.four) 10-4 3M 0.15 0.006 551 9 (1.three 0.6) 10-5 6.3 0.6 and those displaced by protein rotein 4.1 0.2 K30,32N 0.14 0.002 325 4 (three.two 0.7) 10-5 contacts occurring upon RNA binding. ZF1 0.14 0.009 371 four (three.9 1) 10-5 five.0 0.two Since the difference between Gag and -2 7.7 0.5 ZF2 0.15 0.02 208 two (1.6 2) 10 -1 NC binding comes from more CA8.7 0.5 ZF1 + two 0.13 0.03 206 two (1.9 2) 10 CA interactions in Gag, we conclude that H400C 0.14 0.0006 294 three (6.2 six) 10-3 7.9 0.7 6.three 0.4 H421C 0.16 0.02 225 2 (7.five five) 10-4 the CA A dimer interactions are weak H400,421C 0.14 0.02 202 2 (five.4 9) 10-1 9.8 0.7 and mainly nonelectrostatic, consistent with their mostly hydrophobic nature TARPolyA (for evaluation, see Ganser-Pornillos et al. 9.1 0.three WT 0.16 0.008 238 1 (two.2 1) 10-2 2012). -1 10 0.5 WT (750 nM) 0.15 0.02 248 1 (four.four four) 10 -3 It was surprising to locate that eliminatCANC 0.14 0.008 130 2 (3.9 2) 10 4.7 0.3 ing dimerization leads to a significant alter NC 0.049 0.003 122 three (two.three 1) 10-3 3.9 0.three five.0 0.three WM 0.15 0.004 180 three (1.9 1) 10-3 in Gag binding to TARPolyA, as men3M 0.16 0.001 308 1 (4.eight three) 10-3 8.8 0.5 tioned above; WM-Gag includes a substantially K30,32N 0.14 0.007 198 1 (7.0 1) 10-3 eight.0 0.4 reduce Zeff (5) compared with WT Gag -1 ZF1 0.16 0.007 229 1 (6.9 6) 10 ten 0.five (9). Thus, eliminating Gag dimerization ZF2 0.17 0.006 193 1 3.four 4 10 0.5 features a related impact on non-Psi RNA bindZF1 + 2 0.16 0.PMID:23563799 004 202 1 1.7 2 10 0.7 eight.three 0.four H400C 0.13 0.003 205 1 (1.4 0.9) 10-1 ing as deleting MA. Indeed, WM-Gag and H421C 0.17 0.005 212 1 6.1 10 13 0.eight CANC appeared quite related in their H400,421C 0.16 0.006 197 1 8.0 20 11 1 RNA-binding properties (Supplemental C258G Psi Fig. S2). Each proteins bound Psi and TARPolyA with Zeff five, and their Kd(1M) -5 4.7 0.two WT 0.14 0.001 331 four (6.three 2) 10 -5 values have been 10-fold smaller sized to Psi RNA three.3 0.4 NC 0.071 0.006 246 9 (3.3 2) ten than to TARPolyA, reflecting the RNA seZF1 + two 0.14 0.004 227 3 (1.3 2) 10-1 8.eight 0.eight quence specificity with the NC domain. The Psi-12M reduction in optimistic charges interacting 7.1 0.three WT 0.16 0.001 322 five (1.two 0.four) 10-3 with TARPolyA inside the case of WM-Gag suggests that the loss of.