Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a â??visibleâ?? major state and â??invisibleâ?? minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~â??50Â*°C) that are sometimes required to populate protein minor conformers to levels (~â??1%) that can be detected by CEST experiments solvent H/D exchange can lead to â??dipsâ?? in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~â??18Â*kDa T4 Lysozyme (T4L) at 50Â*°C and the ~â??11Â*kDa E. coli hibernation promoting factor (HPF) at 52Â*°C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~â??1â??3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.
Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules
Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules
Abstract
Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in...
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05-24-2018 12:57 AM
Longitudinal relaxation optimized amide 1 H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins
Longitudinal relaxation optimized amide 1 H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins
Abstract
Chemical Exchange Saturation Transfer (CEST) experiments are increasingly used to study slow timescale exchange processes in biomolecules. Although 15N- and 13C-CEST have been the approaches of choice, the development of spin state selective 1H-CEST pulse sequences that separate the effects of chemical and dipolar exchange significantly increases the utility of 1H-based experiments. Pulse schemes have been...
Evaluating the influence of initial magnetization conditions on extracted exchange parameters in NMR relaxation experiments: applications to CPMG and CEST
Evaluating the influence of initial magnetization conditions on extracted exchange parameters in NMR relaxation experiments: applications to CPMG and CEST
Abstract
Transient excursions of native protein states to functionally relevant higher energy conformations often occur on the μsâ??ms timescale. NMR spectroscopy has emerged as an important tool to probe such processes using techniques such as Carrâ??Purcellâ??Meiboomâ??Gill (CPMG) relaxation dispersion and Chemical Exchange Saturation Transfer (CEST). The extraction of kinetic and...
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07-30-2016 04:57 AM
[NMR paper] (13)CHD 2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.
(13)CHD 2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.
Related Articles (13)CHD 2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.
J Biomol NMR. 2015 Aug 14;
Authors: Rennella E, Huang R, Velyvis A, Kay LE
Abstract
An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited...
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08-15-2015 04:01 PM
13 CHD 2 â??CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins
13 CHD 2 â??CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins
Abstract
An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is...
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08-14-2015 10:12 PM
[NMR paper] A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues.
A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues.
Angew Chem Int Ed Engl. 2013 Aug 22;
Authors: Long D, Marshall CB, Bouvignies G, Mazhab-Jafari MT, Smith MJ, Ikura M, Kay LE
Abstract
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09-17-2013 11:36 PM
[NMR paper] A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme der
A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme derivative with an extra cross-link between Glu35 and Trp108--quenching of cooperative fluctuations and effects on the protein stability.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_120x27.gif Related Articles A two-dimensional NMR study of exchange behavior of amide hydrogens in a lysozyme derivative with an extra cross-link between Glu35 and Trp108--quenching of cooperative fluctuations and effects on the protein...
Exchangeable deuterons introduce artifacts in amide 15 N CEST experiments used to study protein conformational exchange
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