Toward Precise Interpretation of DEER-Based Distance Distributions: Insights from Structural Characterization of V1 Spin-Labeled Side Chains

Pulsed electron paramagnetic resonance experiments can measure individual distances between two spin-labeled side chains in proteins in the range of similar to 1.5-8 nm. However, the flexibility of traditional spin-labeled side chains leads to diffuse spin density loci and thus distance distributions with relatively broad peaks, thereby complicating the interpretation of protein conformational states. Here we analyzed the spin-labeled VI side chain, which is internally anchored and hence less flexible. Crystal structures of V1-labeled T4 lysozyme constructs carrying the VI side chain on a-helical segments suggest that VI side chains adopt only a few discrete rotamers. In most cases, only one rotamer is observed at a given site, explaining the frequently observed narrow distance distribution for doubly VI-labeled proteins. We used the present data to derive guidelines that may allow distance interpretation of other V1-labeled proteins for higher-precision structural modeling.