7 research outputs found
Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs
Triarylmethyl (trityl, TAM) based
spin labels represent a promising
alternative to nitroxides for EPR distance measurements in biomolecules.
Herewith, we report synthesis and comparative study of series of model
DNA duplexes, 5′-spin-labeled with TAMs and nitroxides. We
have found that the accuracy (width) of distance distributions obtained
by double electron–electron resonance (DEER/PELDOR) strongly
depends on the type of radical. Replacement of both nitroxides by
TAMs in the same spin-labeled duplex allows narrowing of the distance
distributions by a factor of 3. Replacement of one nitroxide by TAM
(orthogonal labeling) leads to a less pronounced narrowing but at
the same time gains sensitivity in DEER experiment due to efficient
pumping on the narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide
pairs are influenced by the structure of the linker: the use of a
short amine-based linker improves the accuracy by a factor of 2. At
the same time, a negligible dependence on the linker length is found
for the distribution width in TAM/TAM pairs. Molecular dynamics calculations
indicate greater conformational disorder of nitroxide labels compared
to TAM ones, thus rationalizing the experimentally observed trends.
Thereby, we conclude that double spin-labeling using TAMs allows obtaining
narrower spin–spin distance distributions and potentially more
precise distances between labeling sites compared to traditional nitroxides
Physiological-Temperature Distance Measurement in Nucleic Acid using Triarylmethyl-Based Spin Labels and Pulsed Dipolar EPR Spectroscopy
Resolving
the nanometer-scale structure of biomolecules in natural
conditions still remains a challenging task. We report the first distance
measurement in nucleic acid at physiological temperature using electron
paramagnetic resonance (EPR). The model 10-mer DNA duplex has been
labeled with reactive forms of triarylmethyl radicals and then immobilized
on a sorbent in water solution and investigated by double quantum
coherence EPR. We succeeded in development of optimal triarylmethyl-based
labels, approach for site-directed spin labeling and efficient immobilization
procedure that, working together, allowed us to measure as long distances
as ∼4.6 nm with high accuracy at 310 K (37 °C)
A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T 1 relaxation enhancement
Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T(1) relaxation of TAM should enable measurement of interspin distances up to 50 Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins