Simultaneous determination of disulphide bridge topology and three-dimensional structure using ambiguous intersulphur distance restraints: Possibilities and limitations

Knowledge of the native disulphide bridge topology allows the introduction of conformational restraints between remote parts of the peptide chain. Thi s information is therefore of great importance for the successful determina tion of the three-dimensional structure of cysteine-rich proteins by NMR sp ectroscopy. In this paper we investigate the limitations of using ambiguous intersulphur restraints [Nilges, M. (1995) J. Mol. Biol., 245, 645-660] as sociated with NMR experimental information to determine the native disulphi de bridge pattern. Using these restraints in a simulated annealing protocol we have determined the correct topology of numerous examples, including a protein with seven disulphide bridges (phospholipase A(2)) and a protein in which 25% of the total number of residues are cysteines (mu-conotoxin GIII B). We have also characterised the behaviour of the method when only limite d experimental data is available, and find that the proposed protocol permi ts disulphide bridge determination even with a small number of restraints ( around 5 NOEs - including a long-range restraint - per residue). In additio n, we have shown that under these conditions the use of a reduced penalty f unction allows the identification of misassigned NOE restraints. These resu lts indicate that the use of ambiguous intersulphur distances with the prop osed simulated annealing protocol is a general method for the determination of disulphide bridge topology, particularly interesting in the first steps of NMR study of cysteine-rich proteins. Comparison with previously propose d protocols indicates that the presented method is more reliable and the in terpretation of results is straightforward.