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Accurate long‐range distance measurements in a doubly spin‐labeled protein by a four‐pulse, double electron–electron resonance method

Distance determination in disordered systems by a four‐pulse double electron–electron resonance method (DEER or PELDOR) is becoming increasingly popular because long distances (several nanometers) and their distributions can be measured. From the distance distributions eventual heterogeneities and dynamics can be deduced. To make full use of the method, typical distance distributions for structurally well‐defined systems are needed. Here, the structurally well‐characterized protein azurin is investigated by attaching two (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate spin labels (MTSL) by site‐directed mutagenesis. Mutations at the surface sites of the protein Q12, K27, and N42 are combined in the double mutants Q12C/K27C and K27C/N42C. A distance of 4.3 nm is found for Q12C/K27C and 4.6 nm for K27C/N42C. For Q12C/K27C the width of the distribution (0.24 nm) is smaller than for the K27C/N42C mutant (0.36 nm). The shapes of the distributions are close to Gaussian. These distance distributions agree well with those derived from a model to determine the maximally accessible conformational space of the spin‐label linker. Additionally, the expected distribution for the shorter distance variant Q12C/N42C was modeled. The width is larger than the calculated one for Q12C/K27C by 21%, revealing the effect of the different orientation and shorter distance. The widths and the shapes of the distributions are suited as a reference for two unperturbed MTSL labels at structurally well‐defined sites. Copyright © 2008 John Wiley & Sons, Ltd.