25 kcal/mol. This can be one of the possible conformations of the S3b-S4 pair when there is no transmembrane potential (the zero-potential state). Indeed, the crystal structures show the presence of E107 of S3b facing S4 at the vicinity of R123 and H109 is diametrically on the other side.[6�C8] Figure 3 Role of dipolar and side chain charges in the stability of the S3b-S4 pairs. (a) Potential Z-VAD-FMK energy profile of the system of charges on S3b-S4 vs. the relative translational distance between S3b and S4 along the helix axis, in presence (solid symbol) and … On neutralizing E107 of S3b for all possible rotational positions of S4, the total energy becomes positive and the profile almost flattens out [Figure 3a], i.e.
, the net energy of aggregation of S3b-S4 changes from attractive to repulsive, while [Figure 3b] on neutralizing the other charges, the negative (attractive) energy varied. Hence, E107 is the prime candidate in keeping S3b-S4 ��-helices together behaving like a single ��paddle�� unit.[9] Considering the minimum energy profile position at x = 9.0 and �� =120��, the effect of different charges on the stabilization of S3b-S4 pair was studied. Through virtual mutagenesis, on neutralizing each charge one at a time, the energy profile changed [Figure 3b], implying that each charge (dipolar charge or charged residue) has a significant role in stabilization of the S3b-S4 pair. In the absence of the charged residues (R117, R120, R123, R126, and R133), the total energy has increased, and hence the system lost stability, while in the absence of H109, the energy stability was increased.
On neutralization of the C3 and N4 terminal charges, the potential energy profile was raised, while the absence of N3 and C4 charges lowered the minimum energy value. Therefore, the dipolar end charges, in spite of being weaker, have a substantial role in the stability of S3b-S4 helix pair, which is still unexplored experimentally. Figure 3c shows a comparative study of the potential energy contribution of individual charges in stabilizing the S3b-S4 ��-helix pair. Of all the charges, the contribution of the potential energy of Glutamic acid E107 of S3b is maximum, but all other charges also contribute to the stability. In particular, contribution of the dipolar terminal charges C3 is much greater than that of any of the positive Arginine residues of S4, while contribution of N4 is comparable to some of the Arginine residues of S4 ��-helix.
Experimental evidences favor a significant role played by the Arginine residues of S4 in the gating process[5], but the role of the dipolar charges and the Glutamic acid of S3b in KvAP, so far have not been taken into consideration. Our calculations show, however, that in reference to Brefeldin_A the stability of the S3b-S4 pair, the dipolar charges and the negative Glutamic acid cannot be ignored.