An evaluation of least-squares fits to COSY spectra as a means of estimating proton—proton coupling constants II. Applications to polypeptides

Summary

A new computational method for simultaneously estimating all the proton-proton coupling constants in a molecule from COSY spectra [Yang, J.-X. and Havel, T.F. (1994) J. Biomol. NMR, 4, 807–826] is applied to experimental data from two polypeptides. The first of these is a cyclic hexapeptide denoted as VDA (-d-Ala¹-Phe²-Trp³-Lys(Z)⁴-Val⁵-Phe⁶-), in deuterated DMSO, while the second is a 39-residue protein, called decorsin, in aqueous solution. The effect of different data processing strategies and different initial parameter values on the accuracy of the coupling constants was explored. In the case of VDA, most of the coupling constants did not depend strongly on the initial values chosen for the optimization or on how the data were processed. This, together with our previous experience using simulated data, implies strongly that these values are accurate estimates of the coupling constants. They also differ by an average of only 0.36 Hz from the values of the 14 coupling constants that could be measured independently by established methods. In the case of decorsin, many of the coupling constants exhibited a moderate dependence on their initial values and a strong dependence on how the data were processed. With the most successful data processing strategy, the amide-α coupling constants differed by an average of 1.11 Hz from the 21 values that could be measured by established methods, while two thirds of the three-bond coupling constants fell within 1.0 Hz of the ranges obtained by applying the Karplus relation to an independently computed ensemble of distance geometry structures. The averages of the coupling constants over multiple optimizations using random initial values were computed in order to obtain the best possible estimates of the coupling constants. Most clearly incorrect averages can be identified by large standard deviations in the coupling constants or the associated line widths and chemical shifts, and can be explained by strong coupling and/or overlap with the water signal, the diagonal peaks or other cross peaks.