September 2012
Publication year: 2012 Source:Journal of Magnetic Resonance, Volume 222
Modern solid-state NMR methods can acquire high-resolution protein spectra for structure determination. However, these methods use rapid sample spinning and intense decoupling fields that can heat and denature the protein being studied. Here we present a strategy to avoid destroying valuable samples. We advocate first creating a sacrificial sample, which contains unlabeled protein (or no protein) in buffer conditions similar to the intended sample. This sample is then doped with the chemical shift thermometer Sm2Sn2O7. We introduce a pulse scheme called TCUP (for Temperature Calibration Under Pulseload) that can characterize the heating of this sacrificial sample rapidly, under a variety of experimental conditions, and with high temporal resolution. Sample heating is discussed with respect to different instrumental variables such as spinning speed, decoupling strength and duration, and cooling gas flow rate. The effects of different sample preparation variables are also discussed, including ionic strength, the inclusion of cryoprotectants, and the physical state of the sample (i.e. liquid, solid, or slurry). Lastly, we discuss probe detuning as a measure of sample thawing that does not require retuning the probe or using chemical shift thermometer compounds. Use of detuning tests and chemical shift thermometers with representative sample conditions makes it possible to maximize the efficiency of the NMR experiment while retaining a functional sample. Graphical abstract
Highlights
? Heating tests on a sacrificial sample enable efficient NMR of functional proteins. ? Sm2Sn2O7 chemical shift thermometer does not alter freezing point or ionic strength. ? Complete freezing of high salt samples minimizes heating. ? Simple tests for thawing during pulsing are described. ? Increased cooling efficiency is an untapped method for effective heat management.
Many Proteins Exist in a State of 'Disorder' and Yet Are Functional - Science Daily (press release)
Many Proteins Exist in a State of 'Disorder' and Yet Are Functional - Science Daily (press release)
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Many Proteins Exist in a State of 'Disorder' and Yet Are Functional
Science Daily (press release)
When did people realize some proteins violate the rules? It's been about 20 years. The earliest clue was that some protein segments didn't show up in X-ray crystallography or NMR studies, the standard ways of studying protein structure. By the 1990s ...
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10-21-2012 06:59 AM
Heat Management Strategies for Solid-state NMR of Functional Proteins
Heat Management Strategies for Solid-state NMR of Functional Proteins
Publication year: 2012
Source:Journal of Magnetic Resonance</br>
Daniel J. Fowler, Michael J. Harris, Lynmarie K. Thompson</br>
Modern solid-state NMR methods can acquire high-resolution protein spectra for structure determination. However, these methods use rapid sample spinning and intense decoupling fields that can heat and denature the protein being studied. Here we present a strategy to avoid destroying valuable samples. We advocate first creating a sacrificial sample, which contains unlabeled...
nmrlearner
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07-14-2012 01:53 PM
Magic Angle Spinning and Oriented Sample Solid-State NMR Structural Restraints Combine for Influenza A M2 Protein Functional Insights
Magic Angle Spinning and Oriented Sample Solid-State NMR Structural Restraints Combine for Influenza A M2 Protein Functional Insights
Thach V. Can, Mukesh Sharma, Ivan Hung, Peter L. Gor’kov, William W. Brey and Timothy A. Cross
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja3004039/aop/images/medium/ja-2012-004039_0004.gif
Journal of the American Chemical Society
DOI: 10.1021/ja3004039
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/xappAmN-wb8
nmrlearner
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05-25-2012 07:14 PM
Assignment strategies for aliphatic protons in the solid-state in randomly protonated proteins
Assignment strategies for aliphatic protons in the solid-state in randomly protonated proteins
Abstract Biological solid-state nuclear magnetic resonance spectroscopy developed rapidly in the past two decades and emerged as an important tool for structural biology. Resonance assignment is an essential prerequisite for structure determination and the characterization of motional properties of a molecule. Experiments, which rely on carbon or nitrogen detection, suffer, however, from low sensitivity. Recently, we introduced the RAP (Reduced Adjoining Protonation) labeling scheme, which...
Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin [Biophysics and Computational Biology]
Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin
Struts, A. V., Salgado, G. F. J., Brown, M. F....
Date: 2011-05-17
Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state 2H NMR relaxation allows investigation of light-induced changes in local ps–ns time scale motions of retinal bound to rhodopsin. Site-specific 2H labels were introduced into methyl groups of the...
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05-17-2011 08:40 PM
Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin.
Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin.
Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin.
Proc Natl Acad Sci U S A. 2011 Apr 28;
Authors: Struts AV, Salgado GF, Brown MF
Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state (2)H NMR relaxation...
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04-30-2011 12:36 PM
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Cell-free expression and labeling strategies for a new decade in solid-state NMR.
N Biotechnol. 2010 Aug 3;
Authors: Abdine A, Verhoeven MA, Warschawski DE
Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...
Heat management strategies for solid-state NMR of functional proteins
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