Figure 2 Local interaction force between the adjacent termini of S3b-S4 ��-helix pair vs. the distance (��) between the two helices at different angle of rotation selleck chem (��) of S4. (a) At the intracellular side, distance (��) is between N3 … In comparison with the full-length ion channel, in the isolated VSD, according to the structure (PDB 2KYH and 1ORS)[8,9], the extracellular termini experience a net attractive force but at the intracellular end, the net repulsive force (between N3 and R133) (Figure 2a, open symbol) is stronger in nature. This is because the S4 helix in isolated VSD is longer (R117-L148) with C4 terminal 15 residues below R133 and farther away from N3; hence, the attractive force between N3 and C4 becomes weaker and the repulsive force between N3 and R133 predominates, making the bifurcation larger.

In keeping with this finding, the observed distance between the N3 of S3b and R133 of S4 is approximately 26% and 63% greater in the structures of PDB 1ORS and 2KYH, respectively, than in PDB 1ORQ, hence justifying the bifurcation at the intracellular side between the two antiparallel S3b-S4 helices, which is explained by the electrostatic theory. At the termini of S3b-S4 pair of the full-length ion channel, the effect of the primary charges (N3, C4, R133, C3, N4, and R117) involved in these local forces was studied by virtual mutagenesis. (1) On neutralizing all the dipolar charges (N3, C3, N4, and C4), the local force was found to be almost negligible between the termini of the ��-helix pair at both the intracellular end [Figure 2c] and extracellular end [Figure 2d].

(2) When the charged residues (R117 and R133) were mutated, both terminals experienced a comparable local attractive force similar to a typical antiparallel macrodipole pair. Thus, in both cases, the intracellular bifurcation collapsed in absence of the repulsive force. Therefore, the coordinated role of the charged residues and the dipolar charges at the two termini of S3b-S4 pair has a significant effect on the unequal spacing of the mutual spatial position of S3b-S4, i.e., holding the extracellular poles (C3 and N4) closer and the intracellular N3 end far apart from the S4 helix. Role of the charges on the stability of aggregation of S3b-S4 pair The total potential energy (equation 2), of the system of charges of the S3b-S4 pair was computed as a function of the translational motion of S4 along its helical axis and at different angles of rotation (�� = 0��, 60��, 120��, 180��, 200��) [Figure 1d] about its axis.

The energy profiles [Figure 3a] showed minimum energy when the positive residues R117, R120, R123, R126, and R133 face the negative E107 at the translational positions of x = 0, 4.5, 9.0, 13.5, and 24 �� and rotational positions of �� = 0��, 60��, 120��, 180��, and 200��, Brefeldin_A respectively. When S4 was rotated by �� =120�� and translated by x = 9.0 , i.e.