Circular dichroism constrains NMR-derived structures of a folded trinitrophenylated hexapeptide in solution

A trinitrophenylated hexapeptide, with sequence imitating that near the rea ctive lysyl residue (Lys(84) or RLR) in skeletal muscle myosin, has an indu ced circular dichroism (CD) signal in the absorption band: of the trinitrop henyl group (TNP) characteristic to the TNP in a structured environment. Nu clear Overhauser Effect (NOE) and coupling constant data obtained with H-1 NMR confirm that the TNP-hexapeptide (TNP-6p) exists as an ensemble of clos ely related S-dimensional structures. A simulated annealing procedure const rained by the NOE distances produced a solution set of 47 structures for th e TNP-6p with potential energies less than or approximately equal to the ro ot-mean-squared energy fluctuation expected for this peptide. The CD signal s induced in the three lowest-energy electronic transitions of the TNP abso rption bands; were computed for each structure in the solution set using th e matrix method implemented for TNP as the signal donor group. The computed CD signals distinguish two subsets of structures with opposite chirality. One structural isomer subset produces an ensemble averaged CD signal in agr eement with experimental results. The other subset or the total set of stru ctures produce ensemble-averaged CD signals that disagree with the experime ntal results. These findings demonstrate the importance of CD constraints i n the refinement of NMR derived structures of small proteins and peptides a nd that the matrix method is a reliable predictor of CD signals; The TNP-6p can now serve as a practical test case for new theoretical methods for com puting CD signals because of its strong and detailed CD spectrum-and known solution structure.