Studying micro to millisecond protein dynamics using simple amide 15N CEST experiments supplemented with major-state R2 and visible peak-position constraints
Studying micro to millisecond protein dynamics using simple amide 15N CEST experiments supplemented with major-state R2 and visible peak-position constraints
Over the last decade amide 15N CEST experiments have emerged as a popular tool to study protein dynamics that involves exchange between a â??visibleâ?? major state and sparsely populated â??invisibleâ?? minor states. Although initially introduced to study exchange between states that are in slow exchange with each other (typical exchange rates of, 10 to 400Â*sâ??1), they are now used to study interconversion between states on the intermediate to fast exchange timescale while still using low to moderate (5 to 350Â*Hz) â??saturatingâ?? B1 fields. The 15N CEST experiment is very sensitive to exchange as the exchange delay TEX can be quite long (~0.5Â*s) allowing for a large number of exchange events to occur making it a very powerful tool to detect minor sates populated ( \({p}_{minor}\) ) to as low as 1%. When systems are in fast exchange and the 15N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the \({\chi }_{red}^{2}\) versus \({p}_{minor}\) and \({\chi }_{red}^{2}\) versus exchange rate ( \({k}_{ex}\) ) plots can be quite flat with shallow or no minima and the analysis of such 15N CEST data can lead to wrong estimates of the exchange parameters due to the presence of â??spuriousâ?? minima. Here we show that the inclusion of experimentally derived constraints on the intrinsic transverse relaxation rates and the inclusion of visible state peak-positions during the analysis of amide 15N CEST data acquired with moderate B1 values (~50 toâ??~350Â*Hz) results in convincing minima in the \({\chi }_{red}^{2}\) versus \({p}_{minor}\) and the \({\chi }_{red}^{2}\) versus \({k}_{ex}\) plots even when exchange occurs on the 100Â*μs timescale. The utility of this strategy is demonstrated on the fast-folding Bacillus stearothermophilus peripheral subunit binding domain that folds with a rate constantâ??~104Â*sâ??1. Here the analysis of 15N CEST data alone results in \({\chi }_{red}^{2}\) versus \({p}_{minor}\) and \({\chi }_{red}^{2}\) versus \({k}_{ex}\) plots that contain shallow minima, but the inclusion of visible-state peak positions and restraints on the intrinsic transverse relaxation rates of both states during the analysis of the 15N CEST data results in pronounced minima in the \({\chi }_{red}^{2}\) versus \({p}_{minor}\) and \({\chi }_{red}^{2}\) versus \({k}_{ex}\) plots and precise exchange parameters even in the fast exchange regime ( \({k}_{ex}/|\mathrm{\Delta \omega }|\) ~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500Â*sâ??1) from 33.2 to 42.9Â*°C while the unfolding rates (~70 toâ??~500Â*sâ??1) and unfolded state populations (~0.7 toâ??~4.3%) increase with temperature. The results presented here show that protein dynamics occurring on the 10 to 104Â*sâ??1 timescale can be studied using amide 15N CEST experiments.
[NMR paper] ARCHE-NOAH: NMR supersequence with five different CEST experiments for studying protein conformational dynamics
ARCHE-NOAH: NMR supersequence with five different CEST experiments for studying protein conformational dynamics
An NMR NOAH-supersequence is presented consisting of five CEST experiments for studying protein backbone and side-chain dynamics by ^(15)N-CEST, carbonyl-^(13)CO-CEST, aromatic-^(13)C(ar)-CEST, ^(13)C(?)-CEST, and methyl-^(13)C(met)-CEST. The new sequence acquires the data for these experiments in a fraction of the time required for the individual experiments, saving over four days of NMR time per sample.
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nmrlearner
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06-08-2023 09:28 PM
Exchangeable deuterons introduce artifacts in amide 15 N CEST experiments used to study protein conformational exchange
Exchangeable deuterons introduce artifacts in amide 15 N CEST experiments used to study protein conformational exchange
Abstract
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...
[NMR paper] Residue Selective 15N CEST and CPMG Experiments for Studies of Millisecond Timescale Protein Dynamics
Residue Selective 15N CEST and CPMG Experiments for Studies of Millisecond Timescale Protein Dynamics
Publication date: Available online 1 June 2018
Source:Journal of Magnetic Resonance</br>
Author(s): Xiaogang Niu, Jienv Ding, Wenbo Zhang, Qianwen Li, Yunfei Hu, Changwen Jin</br>
Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes...
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06-03-2018 01:00 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...
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03-30-2017 06:42 PM
[NMR paper] Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
From Mendeley Biomolecular NMR group:
Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
Journal of the American Chemical Society (2012). Pramodh Vallurupalli, Guillaume Bouvignies, Lewis E Kay et al.
Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and...
nmrlearner
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10-17-2013 12:49 PM
[NMR paper] Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
From Mendeley Biomolecular NMR group:
Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
Journal of the American Chemical Society (2012). Pramodh Vallurupalli, Guillaume Bouvignies, Lewis E Kay et al.
Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and...
nmrlearner
Journal club
0
11-12-2012 01:53 AM
[NMR paper] Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
From Mendeley Biomolecular NMR group:
Studying "Invisible" Excited Protein States in Slow Exchange with a Major State Conformation.
Journal of the American Chemical Society (2012). Pramodh Vallurupalli, Guillaume Bouvignies, Lewis E Kay et al.
Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and...