ABCMdb

PMID: 22779403

Nadeau VG, Rath A, Deber CM
Sequence hydropathy dominates membrane protein response to detergent solubilization.
Biochemistry. 2012 Aug 7;51(31):6228-37. Epub 2012 Jul 25., [PubMed]

Sentences

No. Mutations Sentence Comment

87 ABCC7 p.Val232Asp ABCC7 p.Glu217Val The intensity of mean residue ellipticity at 222 nm, for example, ranged by ~20 000 deg cm2 dmol-1 among the hairpin sequences (compare E217V and V232D, Figure 2A, left), a result that might not initially be anticipated for a "mild" detergent. Login to comment 89 ABCC7 p.Pro205Ser ABCC7 p.Val232Asp ABCC7 p.Glu217Val ABCC7 p.Ala204Leu ABCC7 p.Glu217Phe We further noted that the spectra of mutant hairpins solubilized in DDM exhibited a gradation of intensities, whereas SDS-solubilized mutant spectra could be divided into "low helicity" (WT, V232D, A204L, and P205S) or "high helicity" (E217V, ES/SE, and E217F) clusters. Login to comment 96 ABCC7 p.Glu217Val ABCC7 p.Glu217Phe The correlation of hydropathy levels in TM3/4 mutants and helicity in both detergents was also apparent in the rank ordering of mutants according to their molar residue ellipticity at 222 nm (Figure 2, right), with the exception of three mutants: E217F, E217V, and ES/SE (Figure 2A). Login to comment 107 ABCC7 p.Val232Asp The WT and mutant sequences complexed with DDM exhibited variable elution volumes, with ES/SE and V232D eluting earliest and latest from the column, respectively (Figure 3B, left). Login to comment 119 ABCC7 p.Val232Asp However, we noted that the elution volumes of the V232D mutant (open circle, Figure 3B) placed this hairpin outside the 95% confidence interval of the line of best fit. Login to comment 120 ABCC7 p.Val232Asp TM3/4-V232D can therefore be considered to differ in its hydrodynamic properties following solubilization by DDM vs SDS. Login to comment 144 ABCC7 p.Pro205Ser ABCC7 p.Glu217Val ABCC7 p.Ala204Leu In DDM (Figure 4B, left), we found that the P205S (E:M = 0.87 ± 0.20), E217V (E:M = 0.78 ± 0.06), E271F (E:M = 0.76 ± 0.12) and A204L spectra (E:M = 0.69 ± 0.02) each displayed excimer fluorescence, and the E:M ratio of each of these "compact" mutants was statistically distinct from the negative control in DDM (E:M = 0.52 ± 0.02, 4.7 × 10-6 ≤ p ≤ 0.008). Login to comment 146 ABCC7 p.Glu217Val ABCC7 p.Ala204Leu In contrast, after SDS solubilization (Figure 4B, right), excimer emission above negative control (E:M = 0.21 ± 0.004) levels was present only in the A204L mutant (E:M = 0.32 ± 0.06, p = 0.027); the E217V mutant had an E:M ratio higher than the negative control, but this difference was of marginal significance (E:M = 0.28 ± 0.04, p = 0.062). Login to comment 165 ABCC7 p.Val232Asp N = 7 for the plots; the line of best fit omits the outlier mutant V232D (open circle, data point outside the 95% confidence interval of the fit). Login to comment 167 ABCC7 p.Val232Asp Note that the line of best fit has a near- unity slope, indicating that the relative order of elution is the same among the hairpin-DDM and hairpin-SDS complexes when V232D is excepted. Login to comment 169 ABCC7 p.Glu217Val A further observation from the sets of CD curves shown in Figure 2 is that the spectra in SDS display ellipticity notably skewed toward the 208 nm lobe, while corresponding spectra in DDM generally have approximately equal ellipticity for the 208 and 222 nm lobes (exception: the loop-based E217V mutant). Login to comment 172 ABCC7 p.Val232Asp Hydropathy Dependence in Local Sequence Context: The Cases of ES/SE and V232D. Login to comment 173 ABCC7 p.Val232Asp The ES/SE-DDM and V232D mutants are distinguished among the groups of DDM- or SDS-solubilized hairpins in that each of these hairpin-detergent complexes exhibits an apparent excluded volume on SEC that is larger than expected based on their rankings in helicity and low E:M ratios (compare Figures 3B, 2B, and 4B). Login to comment 174 ABCC7 p.Val232Asp Because the excluded volume of protein-detergent complexes is not an absolute measure of binding stoichiometry or particle size when particles deviate from compact, spherical shapes, we can only conclude at present that the shapes and/or dynamics of the ES/SE-DDM complex and the V232D-SDS complex differ in some way from the hairpin-DDM and hairpin-SDS complexes of equivalent helicities. Login to comment 189 ABCC7 p.Pro205Ser ABCC7 p.Glu217Val ABCC7 p.Ala204Leu ABCC7 p.Glu217Phe We were thus excited to observe that solubilization by DDM supports a distance <10 Å between pyrene molecules conjugated at the ends of TM3 and TM4, giving rise to a subset of "compact" mutants (P205S, E217V, E217F, and A204L) (Figure 4B, left). Login to comment 193 ABCC7 p.Glu217Val ABCC7 p.Glu217Phe Consistent with this possibility, the "high helicity" group of SDS-solubilized mutants (E217F, E217V, and ES/SE) has E:M ratios that do not exceed the negative control. Login to comment