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Figure 4.
Figure 4. Comparing Interactions in Alternate Kv7 Subtypes
(A) Stoichiometry of coiled-coil assembly domains in all
five Kv7 subtypes shown by Superdex200 (Amersham Biosciences)
size exclusion chromatography. Normalized absorbance is plotted
against elution volume V[E] corrected for void elution volume
V[0] as in Figure 3B. All samples were loaded at a concentration
of 50 μM. Vertically displaced chromatograms show traces for,
from top to bottom, Kv7.1 (black), Kv7.2 (orange), Kv7.3
(purple), Kv7.4 (green), Kv7.5 (pink), and MBP (gray). Vertical
dotted lines indicate the predicted elution volumes of
tetrameric (red) and monomeric (blue) fusion proteins. (Inset)
Standard curve used to calculate molecular weight of eluted
proteins on the Superdex200 column. Molecular weights for each
are as follows (observed ± SD, expected monomer, expected
tetramer); Kv7.1 (180 ± 2 kD, 49.4 kD, 198 kD); Kv7.2
(203 ± 6 kD, 49.3 kD, 197 kD); Kv7.3 (90.3 ± 2 kD,
49.9 kD, 200 kD); Kv7.4 (207 ± 6 kD, 48.8 kD, 195 kD);
Kv7.5 (191 ± 6 kD, 48.9 kD, 196 kD).
(B) Comparative
interaction mapping in all subtypes. Column labels identify
residue types involved in hydrophobic “a” (blue) and “d”
(pink) layer contacts and electrostatic interactions (green)
observed in the Kv7.4 coiled-coil structure. Filled boxes in
table indicate entirely conserved interactions; shaded boxes
indicate nonconserved residues that are still capable of
interacting as predicted; white boxes indicate unfavorable
contacts. Electrostatic interactions involved in networks 1 and
2 are indicated below the alignment.
(C) Stoichiometry of
mutant coiled-coil assembly domains as determined by size
exclusion. Kv7.3 A-domain Tail mutants F622L and D631S/G633E
restore tetramerization. Molecular weights for each are as
follows (observed, expected monomer, expected tetramer); Kv7.3
(90.3 kD, 49.9 kD, 200 kD); Kv7.3 F622L (212 kD, 49.9 kD, 200
kD); Kv7.3 D631S/G633E (208 kD, 49.9 kD, 200 kD). All samples
were loaded onto the column at a concentration of 50 μM.
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