These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels. values) of the His-19 tetrad and found them to be one pH unit lower than the pvalues of the His-37 tetrad in AM2 (18). insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels. values) of the His-19 tetrad and found them to be one pH unit lower than the pvalues of the His-37 tetrad in AM2 (18). This large pchange is surprising, and we hypothesized that the C-terminal His-27 may increase the proton dissociation rate of His-19, thereby stabilizing the neutral state of His-19. If this model were correct, then we would expect His-27 to exhibit higher pvalues than His-19. Although high-resolution structures of the AM2 TM domain have been determined using multiple techniques (8, 9, 11, 12, 14, 16, 22), the structure of the BM2 channel has been much less characterized. To date, there is only a single structure of BM2, solved by solution NMR in DHPC (1,2-dihexanoyl-shows 2D double-quantum (DQ) and single-quantum (SQ) correlation spectra (called INADEQUATE; Ref. 24) of the peptide, measured at pH 5.5 and pH 7.3. With a short dipolar recoupling time of 0.50C0.57 ms for 13C-13C DQ excitation and reconversion, the 2D spectrum exhibits only correlation peaks for directly bonded carbons, thus allowing resonance assignment. Ser-9 shows similar C and C chemical shifts, characteristic of an -helical conformation. Similarly, Ile-14 (64.1 ppm, 36.0 ppm), Leu-18 (55.8 ppm, 40.2 ppm), and Ala-22 (53.4 ppm, 17.0 ppm) adopt -helical C and C chemical shifts, which do not change with pH. Thus, the backbone conformation of the BM2 TM domain is independent of pH and Rabbit Polyclonal to KITH_HHV11 is -helical in the cholesterol-containing membrane, consistent with the result of a previous study using different site-specifically labeled residues (18). Open in a separate window Figure 1. 2D 13C-13C INADEQUATE spectra of membrane-bound BM2(1C33) measured at 243 CP 945598 HCl (Otenabant HCl) K. below). At low pH, two sets of C-C cross-peaks are also resolved but at lower chemical shifts. These peaks can be assigned to cationic His-27 based on their correlations with the C and C2 chemical shifts: the cross-peaks at 57.0 ppm and 25.8 ppm can be assigned to the +4 tetrad (cat4), whereas the 54.2 ppm and 25.1 ppm cross-peaks can be assigned to the +3 tetrad (cat3). Open in a separate window Figure 3. 2D 15N-13C and 13C-13C correlation spectra of His-27 side chain in membrane-bound BM2(1C33). values (25). Fig. 4shows low-temperature 15N spectra as a function of pH. The 250-ppm 15N peak resulted from unprotonated imidazole nitrogen, whereas the 170-ppm peak was due to protonated nitrogens. At high pH, a relatively sharp 15N peak at 167 ppm was detected and can be assigned to tautomer N?2H and tautomer N1H. At the intermediate pH of 6.0, both neutral and cationic NH peaks were observed, whereas below pH 6.0 only cationic NH signals were observed. As expected, the 250-ppm intensity decreased progressively from high to low pH, giving a high [His]/[HisH+] ratio of 7.7 at pH 8.0 but a low ratio of 0.09 at pH 5.5 (Fig. 5, and values of 7.9 0.1, 5.9 0.1, 5.9 0.1, and 5.8 0.1. Thus, the His-27 tetrad already acquired a +1 charge at pH 8, and the +1 charge persisted to pH 5.9, when the other three His-27 residues rapidly protonated to reach the fully charged +4 tetrad (Fig. 5values from the imidazole 15N spectra. is the best fit of the H27 data assuming a single pfor all four histidines. This model does not fit the data well. values. Table 1 pH-dependent His-27 15N peak intensities and neutral-to-cationic CP 945598 HCl (Otenabant HCl) histidine concentration ratios for the pH 5.5 and pH 8.0 samples. Although the showed the full CODEX decay curves, with final is the best-fit assuming a square geometry, whereas show the two distances that provide the best-fit assuming a rectangular geometry. All distance components are Gaussian-weighted (a square model) did not fit the data well (Fig. 6of 6.1 (28), suggesting that His-27 is sufficiently solvent-accessible that it protonates similarly to free histidine in solution. However, the His-27 tetrad differed from free histidine in that it had a high first pof 7.9; in other words, the His-27 tetrad already harbored a +1 charge at neutral CP 945598 HCl (Otenabant HCl) pH. Most importantly, these data indicate that His-27 protonated at 1 pH unit higher (average p= 6.4) than His-19 (average p= 5.1). A previous study measuring pH-dependent Trp fluorescence emission spectra,.
