Exchange enhanced sensitivity gain for solvent-exchangeable protons in 2D 1H–15N heteronuclear correlation spectra acquired with band-selective pulses

Yao, Shenggen; Hinds, Mark G.; Murphy, James M.; Norton, Raymond S.

doi:10.1016/j.jmr.2011.05.013

Cited by 13 publications

(8 citation statements)

References 32 publications

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“…This observation was in agreement with our findings, where these two peaks were not observed in the HSQC spectrum of recombinant 15 N-labelled ShK. However, detection of these peaks in the SOFAST-HMQC spectrum (Fig 5B) suggests that this experiment will be a useful method for recovering some backbone amide cross peaks that are broadened due to intermediate exchange with water (Yao et al, 2011). …”

Section: Discussionsupporting

confidence: 93%

“…Recently, we showed that SOFAST-HMQC spectra have the potential to recover cross peaks from amides in intermediate exchange with water (Yao et al, 2011). As shown in Fig 5B, a SOFAST-HMQC spectrum gave well-defined, intense cross peaks from Cys3 and Met 21, although the peak from Ser2 was still absent.…”

Section: Resultsmentioning

confidence: 99%

“…The amide side chain peaks of Gln16 at 6.25/108 and 6.35/108 ppm are joined by a line. (B) SOFAST-HMQC spectrum of the same sample, showing recovery of the Cys3 and Met21 backbone amide cross peaks (Yao et al, 2011). …”

Section: Figmentioning

confidence: 99%

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Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

Chang

Galea

Leung

et al. 2012

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The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

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Section: Discussionsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

Chang

Galea

Leung

et al. 2012

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“…Consistent with an emerging role for the NusA tag as an alternative to GST fusions to enhance the solubility of poorly-soluble proteins [34,35], we have previously found that the NusA tag was effective in allowing the preparation of mouse IL-3 as a soluble protein from E. coli [18] (Figure 1) with yields and solubility in aqueous solvents sufficient for NMR spectroscopy studies [36–38]. In addition to maintaining hIL-3 in soluble form, the present method allows the preparation of milligram quantities of hIL-3 per litre of shaking E.…”

Section: Discussionmentioning

confidence: 53%

High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

et al. 2013

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Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.

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“…H-15 N HSQC pulse schemes of (A) a standard water-gate sequence, (B) a hemisphere sequence with water-flip back pulses, and (C) a hemisphere sequence with a REBURP pulse. In (C), only the different pulse scheme (shown by square region in (B)) is described.…”

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confidence: 99%

Effects of radiation damping for biomolecular NMR experiments in solution: A hemisphere concept for water suppression

Ishima

2015

Concepts Magnetic Resonance

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Abundant solvent nuclear spins, such as water protons in aqueous solution, cause radiation damping in NMR experiments. It is important to know how the effect of radiation damping appears in high-resolution protein NMR because macromolecular studies always require very high magnetic field strengths with a highly sensitive NMR probe that can easily cause radiation damping. Here, we show the behavior of water magnetization after a pulsed-field gradient (PFG) using nutation experiments at 900 MHz with a cryogenic probe: when water magnetization is located in the upper hemisphere (having +Z component, parallel to the external magnetic field), dephasing of the magnetization by a PFG effectively suppresses residual water magnetization in the transverse plane. In contrast, when magnetization is located in the lower hemisphere (having −Z component), the small residual transverse component remaining after a PFG is still sufficient to induce radiation damping. Based on this observation, we designed 1H-15N HSQC experiments in which water magnetization is maintained in the upper hemisphere, but not necessarily along Z, and compared them with the conventional experiments, in which water magnetization is inverted during the t1 period. The result demonstrates moderate gain of signal-to-noise ratio, 0–28%. Designing the experiments such that water magnetization is maintained in the upper hemisphere allows shorter pulses to be used compared to the complete water flip-back and, thereby, is useful as a building block of protein NMR pulse programs in solution.

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Exchange enhanced sensitivity gain for solvent-exchangeable protons in 2D 1H–15N heteronuclear correlation spectra acquired with band-selective pulses

Cited by 13 publications

References 32 publications

Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

Effects of radiation damping for biomolecular NMR experiments in solution: A hemisphere concept for water suppression

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