Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Enzymol ; 564: 403-25, 2015.
Article in English | MEDLINE | ID: mdl-26477259

ABSTRACT

Pulsed electron paramagnetic resonance (EPR) spectroscopy has become an important tool for structural characterization of biomolecules allowing measurement of the distances between two paramagnetic spin labels attached to a biomolecule in the 2-8 nm range. In this chapter, we will focus on applications of this approach to investigate tertiary structure elements as well as conformational dynamics of nucleic acid molecules. Both aspects take advantage of using specific spin labels that are rigidly attached to the nucleobases, as they allow obtaining not only the distance but also the relative orientation between both nitroxide moieties with high accuracy. Thus, not only the distance but additionally the three Euler angles between both the nitroxide axis systems and the two polar angles of the interconnecting vector with respect to the nitroxide axis systems can be extracted from a single pair of spin labels. To extract all these parameters independently and unambiguously, a set of multifrequency/multifield pulsed EPR experiments have to be performed. We will describe the experimental procedure as well as newly developed spin labels, which are helpful to disentangle all these parameters, and tools which we have developed to analyze such data sets. The procedures and analyses will be illustrated by examples from our laboratory.


Subject(s)
Electron Spin Resonance Spectroscopy/methods , Nucleic Acids/chemistry , Algorithms , Models, Molecular , Nucleic Acid Conformation , Spin Labels
2.
J Am Chem Soc ; 131(17): 6090-2, 2009 May 06.
Article in English | MEDLINE | ID: mdl-19361195

ABSTRACT

Unexpected high DNP enhancements of more than 10 have been achieved in liquid water samples at room temperature and magnetic fields of 9.2 T (corresponding to 400 MHz (1)H NMR frequency and 260 GHz EPR frequency). The liquid samples were polarized in situ using a double-resonance structure, which allows simultaneous excitation of NMR and EPR transitions and achieves significant DNP enhancements at very low incident microwave power of only 45 mW. These results demonstrate the first important step toward the application of DNP to high-resolution NMR, increasing the sensitivity on biomolecules with small sample volumes and at physiologically low concentrations.


Subject(s)
Cyclic N-Oxides/chemistry , Electron Spin Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/methods , Magnetics , Microwaves , Sensitivity and Specificity , Solutions , Spin Labels , Temperature , Water/chemistry
3.
Proc Natl Acad Sci U S A ; 103(36): 13386-90, 2006 Sep 05.
Article in English | MEDLINE | ID: mdl-16938868

ABSTRACT

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439.) in R1 over a distance of 35 A, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y.s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.


Subject(s)
Electron Spin Resonance Spectroscopy , Free Radicals/chemistry , Ribonucleotide Reductases/chemistry , Tyrosine/chemistry , Dimerization , Escherichia coli/enzymology , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Free Radicals/analysis , Kinetics , Models, Chemical , Protein Subunits/chemistry , Ribonucleotide Reductases/classification , Ribonucleotide Reductases/isolation & purification , Ribonucleotide Reductases/metabolism , Solutions/chemistry
4.
Magn Reson Chem ; 43 Spec no.: S248-55, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16235223

ABSTRACT

Within this review, we describe a home-built pulsed electron paramagnetic resonance (EPR) spectrometer operating at 180 GHz as well as the incorporation of two double resonance techniques, electron nuclear double resonance (ENDOR) and pulsed electron double resonance (PELDOR), along with first applications. Hahn-echo decays on a TEMPO/polystyrene sample are presented, demonstrating that the observation of anisotropic librational motions is possible in a very precise manner at high magnetic fields. Bisdiphenylene-phenyl-allyl is used as a model system to illustrate the performance of the setup for 1H-ENDOR using the Mims as well as the Davies sequence. Furthermore, first 1H-Mims and Davies ENDOR spectra on a biological sample, the wild-type Ras*Mn2+*GDP protein, are reported. The capability of the 180-GHz PELDOR setup is demonstrated using the three-pulse ELDOR sequence on the protein ribonucleotide reductase (RNR) subunit R2 from Escherichia coli, which contains two tyrosyl radicals at a 33 angstroms distance. At 180 GHz, orientation selectivity is observed and the modulation frequency is found to be in good agreement with theoretical predictions.


Subject(s)
Electron Spin Resonance Spectroscopy/instrumentation , Electron Spin Resonance Spectroscopy/methods , Cyclic N-Oxides/chemistry , Escherichia coli/enzymology , Free Radicals/chemistry , Free Radicals/metabolism , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Manganese/chemistry , Manganese/metabolism , Polystyrenes/chemistry , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolism , Tyrosine/chemistry , Tyrosine/metabolism , ras Proteins/chemistry , ras Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...