Long-Range Distance Measurements in Proteins at Physiological Temperatures Using Saturation Recovery EPR Spectroscopy

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• 作者：Zhongyu Yang ; Gonzalo Jim茅nez-Os茅s ; Carlos J. L贸pez ; Michael D. Bridges ; K. N. Houk ; Wayne L. Hubbell
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：October 29, 2014
• 年：2014
• 卷：136
• 期：43
• 页码：15356-15365
• 全文大小：511K
• ISSN：1520-5126

文摘

Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron鈥揺lectron resonance (DEER), is carried out at cryogenic temperatures (50鈥?0 K) to increase the short spin鈥搒pin relaxation time (T₂) upon which the technique relies. A challenge is to measure long-range distances (20鈥?0 脜) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin鈥搇attice relaxation rate (T₁^鈥?) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu²⁺, it has been found that interspin distances 鈮?5 脜 can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to 鈮?0 脜 using spin labels with long T₁, a high-affinity 5-residue Cu²⁺ binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu²⁺ electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu²⁺ loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu²⁺/nitroxide DEER at cryogenic temperatures and T₁ relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.