15N- and 2H-substituted maleimide spin labels: Improved sensitivity and resolution for biological EPR studies
AUTOR(ES)
Beth, A. H.
RESUMO
The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of 15N for 14N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [2H]MSL and the 15N-substituted and deuterated derivative [15N, 2H]MSL were compared with those of the parent MSL. The [15N, 2H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (i) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (ii) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing 14N with 15N. In the freely tumbling state, the spectra of [15N, 2H]MSL and [2H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [15N, 2H]MSL and [2H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [15N, 2H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 μsec to 1 msec. The EPR spectrum of [15N, 2H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and g tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the 15N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by 15N substitution. Use of 15N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=319926Documentos Relacionados
- Structural and motional changes in glyceraldehyde-3-phosphate dehydrogenase upon binding to the band-3 protein of the erythrocyte membrane examined with [15N,2H]maleimide spin label and electron paramagnetic resonance.
- Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies.
- Canada's nutrition labels: a new world standard?
- A NITROXIDE-MALEIMIDE SPIN LABEL*
- Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 Å resolution