MtsslWizard: In Silico Spin-Labeling and Generation of Distance Distributions in PyMOL

MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity

Pulse dipolar electron paramagnetic resonance spectroscopy distance measurements at low nanomolar concentrations : the CuII-trityl case

Funding: To meet institutional and research funder open access requirements, any accepted manuscript arising shall be open access under a Creative Commons Attribution (CC BY) reuse licence with zero embargo. The authors acknowledge support by a University of St Andrews-University of Bonn Collaborative Research Grant, by the Wellcome Trust (204821/Z/16/Z), and by the EPSRC (EP/X016455/1). B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). O.S. thanks the DFG for funding (420322655). C.A.H. thanks the DAAD for a travel and research scholarship. The authors thank the StAnD (St Andrews and Dundee) EPR grouping for long-standing support and the St Andrews mass spectrometry and proteomics facility for equipment access.Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using CuII ions and trityl yields a more than 3-fold sensitivity increase compared to the established CuII-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.Publisher PDFPeer reviewe

Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe Ç

Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies

Base-specific spin-labeling of RNA for structure determination

To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5-iodouridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE (R) chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed

MAPPING GLOBAL FOLDS OF OLIGONUCLEOTIDES BY PULSED ELECTRON-ELECTRON DOUBLE RESONANCE

The understanding of structure-dynamics-function relationships in oligonucleotides or oligonucleotide/protein complexes calls for biophysical methods that can resolve the structure and dynamics of such systems on the critical nanometer length scale. A modern electron paramagnetic resonance (EPR) method called pulsed electron-electron double resonance (PELDOR or DEER) has been shown to reliably and precisely provide distances and distance distributions in the range of 1.5-8 nm. In addition, recent experiments proved that a PELDOR experiment also contains information on the orientation of labels, enables easy separation of coupling mechanisms and allows for counting the number of monomers in complexes. This chapter briefly summarizes the theory, describes how to perform and analyze such experiments and discusses the limitations.</p

MAPPING GLOBAL FOLDS OF OLIGONUCLEOTIDES BY PULSED ELECTRON-ELECTRON DOUBLE RESONANCE

The understanding of structure-dynamics-function relationships in oligonucleotides or oligonucleotide/protein complexes calls for biophysical methods that can resolve the structure and dynamics of such systems on the critical nanometer length scale. A modern electron paramagnetic resonance (EPR) method called pulsed electron-electron double resonance (PELDOR or DEER) has been shown to reliably and precisely provide distances and distance distributions in the range of 1.5-8 nm. In addition, recent experiments proved that a PELDOR experiment also contains information on the orientation of labels, enables easy separation of coupling mechanisms and allows for counting the number of monomers in complexes. This chapter briefly summarizes the theory, describes how to perform and analyze such experiments and discusses the limitations.</p