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Abstract

We present an evaluation of the accuracy and precision of relaxation rates calculated using a variety of methods, applied to data sets obtained for several very different protein systems. We show that common methods of data evaluation, such as the determination of peak heights and peak volumes, may be subject to bias, giving incorrect values for quantities such as R1 and R2. For example, one common method of peak-height determination, using a search routine to obtain the peak-height maximum in successive spectra, may be a source of significant systematic error in the relaxation rate. The alternative use of peak volumes or of a fixed coordinate position for the peak height in successive spectra gives more accurate results, particularly in cases where the signal/noise is low, but these methods have inherent problems of their own. For example, volumes are difficult to quantitate for overlapped peaks. We show that with any method of sampling the peak intensity, the choice of a 2- or 3-parameter equation to fit the exponential relaxation decay curves can dramatically affect both the accuracy and precision of the calculated relaxation rates. In general, a 2-parameter fit of relaxation decay curves is preferable. However, for very low intensity peaks a 3 parameter fit may be more appropriate.

Treatment of Peak Intensity Uncertainties in NMR Relaxation Data Analysis Can Lead to Severe Artifacts

“…NMR-experiments for determining the longitudinal and transversal 15 N relaxation rates were recorded on a Bruker Avance DRX 600 spectrometer at 298 K in 5 mM ammonium acetate buffer, 25 mM calcium chloride, 0.02% sodium azide, and 10% D 2 O at pH 6 (uncorrected meter reading) with a protein concentration of 1.5 mM. For R 1 thirteen data points' rates were recorded with delays of 6.9 (2x), 28.3, 60.5, 124.9 (2x), 253.6, 500.3, 752.4, 1004.4 (2x), 1497.8, and 2001.9 ms for the conventional sampling scheme.…”

“…N Relaxation, which is routinely used to probe backbone-amide dynamics, is experimentally measured via the longitudinal and transversal relaxation rates and the heteronuclear nuclear Overhauser effect (NOE) [6,11,12]. Rates are obtained by fitting mono-exponential decays to signal intensities extracted from a series of [ 1 H- 15 N]-HSQC type spectra recorded with different delays during evolution to allow for relaxation [11,12]. For accurate and precise rates and rate uncertainties it is crucial to maintain stable experimental conditions throughout the experiments [12][13][14].…”

“…Experimental data, however, can be considerably influenced by the way the data are treated. In a recent study Viles et al have pointed out that the method of peak height extraction and the choice of the parametrization for the fitting procedure affect both, accuracy and precision of the rates obtained [15].…”

Quantitative Analysis of Biomolecular NMR Spectra: A Prerequisite for the Determination of the Structure and Dynamics of Biomolecules

“…The propagation of the experimental errors can be assessed using a graphical method [253], or by simulations on synthetic data [254]. The influence of the spectral peak-fitting method on the value of the relaxation rates was also studied [255]. An automatic analysis method is used [256] to avoid user-dependent biases in the choice of a motion model.…”

Applications of NMR Spin Relaxation Methods for Measuring Biological Motions

“…This determination is straightforward: The duration of the CPMG period (at a single value of τ cp ) is varied with time and the resulting crosspeak volumes fit to an exponential decay with a rate constant defined as R 2 using a nonlinear least-squares routine. 97,98 The uncertainties in the rate constants should be determined as discussed by Mandel et al 99 The number of time points necessary to fully define the exponential decay of transverse signal is an important consideration that has been discussed elsewhere. 78,81,98,99 An important aspect of this consideration is to collect more data at the shorter and longer time points as these data are more useful in restraining the fit.…”