CONFORMATIONAL VARIABILITY OF SOLUTION NUCLEAR-MAGNETIC-RESONANCE STRUCTURES

In structure determination by X-ray crystallography and solution NMR s pectroscopy experimental data are collected as time and ensemble-avera ges. Thus, in principle, appropriate time and ensemble-averaged models should be used. Refinement of: an ensemble of conformers rather than one single structure against the experimental NMR data could, however, result in overfitting the data because of the significantly increased number of parameters. To avoid overfitting, complete cross-validation , which provides an unbiased measure of the fit, has been applied to n uclear Overhauser effect derived distance refinement. Using two synthe tic test cases, a correlation was demonstrated between the cross-valid ated measure to the fit (defined in terms of root-mean-square deviatio ns from the distance restraints and number of violations) and the numb er of models that best reproduce the conformational variability in sol ution. A new method, based on a probability map, has been used to gene rate good representations of the resulting ensembles of structures. Th e method has also been applied to observed NMR data for two proteins, interleukin 4 and interleukin 8. For interleukin 4, cross-validation i ndicates that a single-conformer model gives the most accurate represe ntation of the structure, whereas conventional measures of fit between the experimental data and those calculated from the model decrease wh en increasing the number of conformers, indicating overfitting. For in terleukin 8, complete cross-validation predicts a twin-conformer model to be the most faithful representation of the experimental data. Two distinct conformations for the loop formed by residues 16 to 22 emerge from the family of twin-conformer structures. The putative alternate conformation of the loop is not observed in the crystal structure of i nterleukin 8. However, because of crystal packing contacts in this reg ion this does not necessarily exclude the presence of the alternate co nformation in solution. The twin-conformer model is supported by obser ved chemical exchange line broadening for the amide of His18 obtained by N-15 relaxation studies. This region has also been implied to be in volved in receptor binding.