Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd3+ spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and τC14, labeled at strategically selected sites with either two nitroxides or two Gd3+ spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd3+ tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (∼9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd3+-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd3+-Gd3+ distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein τC14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd3+-Gd3+ distance distribution with a maximum at 3.4 nm. The Gd3+-Gd3+ distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd3+ labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection. © 2010 American Chemical Society.
Life Sciences Commons, Physical Sciences and Mathematics Commons, Social and Behavioral Sciences Commons
Potapov, A., Yagi, H., Huber, T., Jergic, S., Dixon, N. E., Otting, G. & Goldfarb, D. (2010). Nanometer-scale distance measurements in proteins using Gd3+ spin labeling. Journal of the American Chemical Society, 132 (26), 9040-9048.