Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: a demonstration of the connectivity tracing assignment tools (CONTRAST) software package.

The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package. The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins. The program's performance is demonstrated here by its analysis of realistic computer-generated data for IIIGlc, a 168-residue signal-transducing protein of Escherichia coli [Pelton et al. (1991) Biochemistry, 30, 10043-10057]. Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested. The algorithm produces sequential assignments from appropriate peak lists of nD NMR data. It quantifies the ambiguity of each assignment and provides ranked alternatives. A 'best first' approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments. The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in user-defined tolerances on the performance of the algorithm.