The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.
[Question from NMRWiki Q&A forum] High Pressure NMR Samples
High Pressure NMR Samples
I need to run NMR on samples of anhydrous ammonia. The boiling point of anhydrous ammonia is -33.34 deg C. In order to be able to have enough time to acquire spectra, I would like to seal the NMR tubes. Once the sample tubes are sealed, the ammonia will build up pressure until it reaches equilibrium and exists as a liquid at room temperature (at approximately 10 atm of pressure). I know NMR tubes exist which can easily withstand these sorts of pressures, but my main problem is in finding a way to seal the tube. I have tried sealing the tubes by melting the...
nmrlearner
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02-23-2012 09:08 PM
[Question from NMRWiki Q&A forum] optimization of mixing time 3D NOESY experments
optimization of mixing time 3D NOESY experments
Could you please explain that how to optimize mixing time for 3D NOESY experiments for protein like C13 edit NOESY and N15 EDIT NOESY and i want keep spin diffusion as much as less ?
Check if somebody has answered this question on NMRWiki QA forum
nmrlearner
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09-21-2011 05:25 AM
DNP by Thermal Mixing under Optimized Conditions Yields >60 000-fold Enhancement of 89Y NMR Signal
DNP by Thermal Mixing under Optimized Conditions Yields >60 000-fold Enhancement of 89Y NMR Signal
Lloyd Lumata, Ashish K. Jindal, Matthew E. Merritt, Craig R. Malloy, A. Dean Sherry and Zoltan Kovacs
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja201880y/aop/images/medium/ja-2011-01880y_0010.gif
Journal of the American Chemical Society
DOI: 10.1021/ja201880y
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/lZjUD7bs_fI
nmrlearner
Journal club
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05-13-2011 07:49 PM
The NMR Structure of FliK, the Trigger for the Switch of Substrate Specificity in the Flagellar Type III Secretion Apparatus.
The NMR Structure of FliK, the Trigger for the Switch of Substrate Specificity in the Flagellar Type III Secretion Apparatus.
The NMR Structure of FliK, the Trigger for the Switch of Substrate Specificity in the Flagellar Type III Secretion Apparatus.
J Mol Biol. 2011 Apr 12;
Authors: Mizuno S, Amida H, Kobayashi N, Aizawa SI, Tate SI
The flagellar cytoplasmic protein FliK controls hook elongation by two successive events: by determining hook length and by stopping the supply of hook protein. These two distinct roles are assigned to different...
nmrlearner
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04-25-2011 11:53 AM
[NMR paper] Two structural subdomains of barstar detected by rapid mixing NMR measurement of amid
Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange.
Related Articles Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange.
Proteins. 1998 Feb 15;30(3):295-308
Authors: Bhuyan AK, Udgaonkar JB
Equilibrium amide hydrogen exchange studies of barstar have been carried out at pH 6.7, 32 degrees C using one- and two-dimensional nuclear magnetic resonance. An unusually large fraction of the backbone amide hydrogens of barstar exchange too fast to...
nmrlearner
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11-17-2010 11:06 PM
Homonuclear Mixing Sequences for Perdeuterated Proteins
Homonuclear Mixing Sequences for Perdeuterated Proteins
Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 26 October 2010</br>
Kuo-Ying, Huang , Ansgar B., Siemer , Ann E., McDermott</br>
We test the performance of several 13C homonuclear mixing sequences on perdeuterated microcrystalline ubiquitin. All sequences were applied without 1H decoupling and at relatively low MAS frequencies. We found that RFDR gave the highest overall transfer efficiency and that DREAM performs surprisingly well under these conditions being twice...
Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes
Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes
Anika Kirschstein, Christian Herbst, Kerstin Riedel, Michela Carella, Jörg Leppert, Oliver Ohlenschläger, Matthias Görlach and Ramadurai Ramachandran
Journal of Biomolecular NMR; 2008; 40(4); pp 227-237
Abstract:
We have explored the design of broadband scalar coupling mediated 13C–13C and cross-relaxation suppressed 1H–1H TOCSY sequences employing phase/amplitude modulated inversion pulses. Considering a variety of supercycles, pulsewidths and a RF field strength of 10 kHz, the Fourier coefficients...
Mixing apparatus for preparing NMR samples under pressure.
Linear Mode
