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

Abstract

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.



Source: Journal of Biomolecular NMR