FIGURE 3.

Detection of the intramolecular spin-spin interactions by the DEER (or CW) experiment. A, rationale for the intra-molecular spin-spin interaction by DEER measurement. Left panel, in the presence of 6-fold excess of unlabeled BAK (sBAK/C154S-ΔC-His) protein (black dots), doubly labeled BAK protein (red dot) forms pores in the membrane by the activation with p7/p15 Bid. Only the intramolecular spin-spin interactions between the two nitroxide spin labels (XR1-YR1) will be observed by the DEER approach because the distance between the doubly labeled BAK proteins increase beyond the detection limit. Right panel, a mixture of two singly spin-labeled BAK proteins (green dots) is used in the presence of unlabeled BAK (sBAK/C154S-ΔC-His) at the indicated ratio to form pores. DEER modulations will not be observed in the mixture of two singly labeled proteins due to an increase in the inter-spin distance by dilution with the unlabeled proteins. A CW experiment can also be performed this way. B, DEER data for the doubly spin-labeled proteins and a mixture of two singly labeled proteins in the presence of excess unlabeled proteins. The DEER modulation curves for the indicated membrane-inserted BAK samples prepared with the doubly labeled BAK or the mixture of two singly labeled proteins are shown in red and green traces, respectively. Their two-dimensional (left panels) or three-dimensional (right panels) background signals fitted are shown in black solid lines (for the doubly labeled sample) or black dotted lines (for the mixture of two singly labeled BAK).