Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A spirocyclohexyl nitroxide amino acid spin label for pulsed EPR spectroscopy distance measurements.

ABSTRACT: Site-directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide alpha-amino acid and its N-(9-fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin-echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.

SUBMITTER: Rajca A

PROVIDER: S-EPMC3103209 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A spirocyclohexyl nitroxide amino acid spin label for pulsed EPR spectroscopy distance measurements.

Rajca Andrzej A Kathirvelu Velavan V Roy Sandip K SK Pink Maren M Rajca Suchada S Sarkar Santanu S Eaton Sandra S SS Eaton Gareth R GR

Chemistry (Weinheim an der Bergstrasse, Germany) 20100501 19

Site-directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide alpha-amino acid and its N-(9-fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin-echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatu ...[more]

PMID: 20391558

Similar Datasets

The spatial effect of protein deuteration on nitroxide spin-label relaxation: implications for EPR distance measurement.

Project description:Pulsed electron-electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of bio-macromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50 K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (Tm). It has become normal practice to use deuterated solvents to extend Tm and recently it has been demonstrated that deuteration of the underlying protein significantly extends Tm. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.

| S-EPMC4245719 | biostudies-literature

Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution.

Project description:Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.

| S-EPMC3409244 | biostudies-literature

High-resolution EPR distance measurements on RNA and DNA with the non-covalent G spin label.

Project description:Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

| S-EPMC6954412 | biostudies-literature

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy.

Project description:The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.

| S-EPMC8706713 | biostudies-literature

Long-range distance determination in fully deuterated RNA with pulsed EPR spectroscopy.

Project description:Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is powerful in structure and dynamics study of biological macromolecules by providing distance distribution information ranging from 1.8 to 6 nm, providing that the biomolecules are site-specifically labeled with paramagnetic tags. However, long distances up to 16 nm have been measured on perdeuterated and spin-labeled proteins in deuterated solvent by PELDOR. Here we demonstrate long-range distance measurement on a large RNA, the 97-nucleotide 3'SL RNA element of the Dengue virus 2 genome, by combining a posttranscriptional site-directed spin labeling method using an unnatural basepair system with RNA perdeuteration by enzymatic synthesis using deuterated nucleotides. The perdeuteration removes the coupling of the electron spins of the nitroxide spin labels from the proton nuclear spin system of the RNA and does extend the observation time windows of PELDOR up to 50 μs. This enables one to determine long distances up to 14 nm for large RNAs and their conformational flexibility.

| S-EPMC8758415 | biostudies-literature

Orthogonal spin labeling and Gd(III)-nitroxide distance measurements on bacteriophage T4-lysozyme.

Project description:We present the first example of chemoselective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a gadolinium(III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

| S-EPMC3634801 | biostudies-literature

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Project description:Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2'dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

| S-EPMC7229862 | biostudies-literature

EPR relaxation-enhancement-based distance measurements on orthogonally spin-labeled T4-lysozyme.

Project description:Lanthanide-induced enhancement of the longitudinal relaxation of nitroxide radicals in combination with orthogonal site-directed spin labeling is presented as a systematic distance measurement method intended for studies of bio-macromolecules and bio-macromolecular complexes. The approach is tested on a water-soluble protein (T4-lysozyme) for two different commercially available lanthanide labels, and complemented by previously reported data on a membrane-inserted polypeptide. Single temperature measurements are shown to be sufficient for reliable distance determination, with an upper measurable distance limit of about 5-6 nm. The extracted averaged distances represent the closest approach in Ln(III) -nitroxide distance distributions. Studies of conformational changes and of bio-macromolecule association-dissociation are proposed as possible application area of the relaxation-enhancement-based distance measurements.

| S-EPMC3804414 | biostudies-literature

Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers.

Project description:This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at K(a) band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59±12Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60Å, and analysis indicates that distance measurements up to 100Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

| S-EPMC3081411 | biostudies-literature

Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label.

Project description:We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

| S-EPMC6702148 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data