Distance distribution
information obtained by pulsed dipolar EPR
spectroscopy provides an important contribution to many studies in
structural biology. Increasingly, such information is used in integrative
structural modeling, where it delivers unique restraints on the width
of conformational ensembles. In order to ensure reliability of the
structural models and of biological conclusions, we herein define
quality standards for sample preparation and characterization, for
measurements of distributed dipole–dipole couplings between
paramagnetic labels, for conversion of the primary time-domain data
into distance distributions, for interpreting these distributions,
and for reporting results. These guidelines are substantiated by a
multi-laboratory benchmark study and by analysis of data sets with
known distance distribution ground truth. The study and the guidelines
focus on proteins labeled with nitroxides and on double electron–electron
resonance (DEER aka PELDOR) measurements and provide suggestions on
how to proceed analogously in other cases.
Distance learning: Under physiological conditions telomeric sequences adopt different quadruplex topologies, although the exact nature of these species is currently debated. Double spin‐labeled oligonucleotides consisting of guanosine‐rich telomeric repeats are synthesized and characterized by pulsed EPR techniques. In solution in the presence of K+ ions the propeller and basket quadruplex conformations co‐exist as a 1:1 mixture.
Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis-Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.
DEER strides: The development of analytical tools for elucidating native structures inside cells is an ongoing challenge. Conformational changes of DNA upon injection into living cells (see figure) can be detected by a combination of site‐directed spin labeling and DEER (double electron–electron resonance) spectroscopy. This technique offers structural information on the nm scale, in cellulo, background‐free and at low concentrations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.