RESUMEN
The investigation of the structure and conformational dynamics of biomolecules under physiological conditions is challenging for structural biology. Although pulsed electron paramagnetic resonance (like PELDOR) techniques provide long-range distance and orientation information with high accuracy, such studies are usually performed at cryogenic temperatures. At room temperature (RT) PELDOR studies are seemingly impossible due to short electronic relaxation times and loss of dipolar interactions through rotational averaging. We incorporated the rigid nitroxide spin label Ç into a DNA duplex and immobilized the sample on a solid support to overcome this limitation. This enabled orientation-selective PELDOR measurements at RT. A comparison with data recorded at 50â K revealed averaging of internal dynamics, which occur on the ns time range at RT. Thus, our approach adds a new method to study structural and dynamical processes at physiological temperature in the <10â µs time range with atomistic resolution.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Ácidos Nucleicos/química , Simulación de Dinámica Molecular , Óxido Nítrico/química , Conformación de Ácido Nucleico , Marcadores de Spin , TemperaturaRESUMEN
An isoindoline-nitroxide derivative of guanine (Ç´, "G-spin") was shown to bind specifically and effectively to abasic sites in duplex RNAs. Distance measurements on a Ç´-labeled duplex RNA with PELDOR (DEER) showed a strong orientation dependence. Thus, Ç´ is a readily synthesized, orientation-selective spin label for "mix and measure" PELDOR experiments.
Asunto(s)
Óxidos de Nitrógeno/química , ARN/química , Coloración y Etiquetado/métodos , Secuencia de Bases , Espectroscopía de Resonancia por Spin del Electrón , Guanina/química , Indoles/química , Estructura MolecularRESUMEN
We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1ß (IL1ß) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1ß-S2R2 and the respective single-loop-LBT constructs IL1ß-S2, IL1ß-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd(3+) as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.