FIGURE 5.

Proximity of helix α5 residues in membrane-inserted BAK oligomers. A, spin dilution experiment for helix α5 residues. The membrane-inserted BAK samples were prepared with the indicated spin-labeled proteins in the presence (3:4) or absence (7:0) of sBAK/C154S-ΔC-His at the indicated ratio as described under “Experimental Procedures.” The area-normalized EPR spectra of the spin-diluted samples (3:4 mixture, black trace) of the indicated membrane-inserted BAK are superimposed to those of the undiluted samples (7:0 mixture, red trace). The ratios of the amplitude of the central line for the 7:0 mixture (h₀) to that for 3:4 mixture (h_d) are indicated. The red arrows for 128R1 indicate the splitting of the EPR lines in the absence of the spin dilution (7:0 mixture) due to the strong spin-spin interactions between two 128R1 residues in close proximity in the membrane-inserted state of BAK (see also Fig. 5C). The hyperfine extrema of the spectrum for the spin-diluted sample (3:4 mixture) are denoted by “im,” which indicate a severely restricted tumbling motion (i.e. immobile) in this spin label. B, distances of 124R1, 142R1, and 143R1 in membrane-inserted BAK to their nearest neighbors estimated by the CW deconvolution method. The CW deconvolution method (23) was applied to the spectra for the indicated mutants (red traces, left panel), resulting in simulated fits (black dotted lines on the left panel) superimposed to the spectra for 7:0 mixture of the indicated residues. The corresponding distance distribution functions are shown on the right panel. C, direct spectral simulation of perdeuterated spin label R1-d15 at residue 128 (128R1-d15) for inter-spin distance estimation. The EPR spectra of the BAK Cys-128 mutant labeled with a perdeuterated spin label (R1-d15, top panel) were obtained in the presence or absence of sBAK/C154S-ΔC-His in membrane at the indicated ratios (dotted traces) at −30 °C. The spectrum for the 1:6 mixture was used to calculate the line width and the principal elements of the g and A tensors of the spin label, which gave the corresponding fit (middle panel, red trace). The spectrum from the 7:0 mixture was fitted theoretically with these spectral parameters for the two C2-symmetry-related nitroxides that are oriented relative to each other as defined by the Euler angles α, β, and γ as described under “Experimental Procedures” (29–31). This resulted in eight sets of symmetry-equivalent Euler angles that fit the data best, all yielding an identical inter-spin distance of 14.3 Å. Only one set of Euler angles is shown here. D, DEER data for membrane-inserted BAK in the absence of spin dilution. DEER experiments were carried out without spin dilution with the indicated membrane-inserted BAK proteins. The DEER data (left column) were analyzed by the DeerAnalysis program using Tikhonov regularization, resulting in distance distribution functions on the right panel (red traces), which could be best fitted with two Gaussian models (blue traces, right panel) with the average inter-spin distance <r> and the standard deviation for the shorter distances indicated. The dotted lines superimposed to the DEER signals are the calculated DEER signal for the distance fit. E, angular clustering of certain residues in helix α5. Side chains of residues Ser-122, Gly-124, Ala-128, Arg-135, Gln-142, and Arg-143, which show spin-spin interactions for the corresponding R1 residues, are clustered on one side of helix α5. Ser-122 is in the loop just upstream of α5 N terminus. F, anti-parallel arrangement of two α5 helices in the BAK homodimer. The C_α carbon atoms of the three residues indicated are shown in spheres on α5 helices along with the inter-spin distances for the symmetry-related spin label pairs in the BAK homology model of BAX BH3-in-groove homodimer model (6).