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1.
Proc Natl Acad Sci U S A ; 113(33): 9187-92, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27489348

RESUMO

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Dobramento de Proteína , Prótons
2.
J Biomol NMR ; 62(3): 253-61, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26078089

RESUMO

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Prótons
3.
J Biomol NMR ; 54(2): 123-7, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22872367

RESUMO

We have recently proposed sedimented solute NMR (SedNMR) as a solid-state method to access biomolecules without the need of crystallization or other sample manipulation. The drawback of SedNMR is that samples are intrinsically diluted and this is detrimental for the signal intensity. Ultracentrifugal devices can be used to increase the amount of sample inside the rotor, overcoming the intrinsic sensitivity limitation of the method. We designed two different devices and we here report the directions for using such devices and the relevant equations for determining the parameters for sedimentation.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Ultracentrifugação/métodos , Cristalização , Soluções
4.
J Magn Reson ; 156(1): 131-7, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12081450

RESUMO

The 2D satellite transition magic angle spinning (STMAS) experiment generates efficiently high-resolution isotropic NMR spectra of half-integer quadrupolar nuclei. The experiment involves excitation and coherence transfer of satellite transitions into the central transition. It requires efficient refocusing of satellite transitions and sample spinning at a very accurate magic angle to cancel the first-order quadrupolar interaction effect. A review of all parameters relevant to optimizing the STMAS experiment is presented, including pulse sequence calibration, regulating spinning speed, magic angle adjustment, optimization of satellite transition excitation, and coherence transfer for both I = 3/2 and I > or =5/2 nuclei.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Sulfatos/química , Calibragem
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