Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling

详细信息    查看全文

• 作者：Alexey Potapov ; Hiromasa Yagi ; Thomas Huber ; Slobodan Jergic ; Nicholas E. Dixon ; Gottfried Otting ; Daniella Goldfarb
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：July 7, 2010
• 年：2010
• 卷：132
• 期：26
• 页码：9040-9048
• 全文大小：358K
• 年卷期：v.132,no.26(July 7, 2010)
• ISSN：1520-5126

文摘

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 τ_C14, 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 τ_C14, 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.