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

A trinitrophenylated hexapeptide, with sequence imitating that near the reactive lysyl residue (Lys⁸⁴ or RLR) in skeletal muscle-myosin, has an induced circular dichroism (CD) signal in the absorption band of the trinitrophenyl group (TNP) characteristic to the TNP in a structured environment. Nuclear Overhauser Effect (NOE) and coupling constant data obtained with ¹H NMR confirm that the TNP-hexapeptide (TNP-6p) exists as an ensemble of closely related 3-dimensional structures. A simulated annealing procedure constrained by the NOE distances produced a solution set of 47 structures for the TNP-6p with potential energies less than or approximately equal to the root-mean-squared energy fluctuation expected for this peptide. The CD signals induced in the three lowest-energy electronic transitions of the TNP absorption bands were computed for each structure in the solution set using the 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 agreement with experimental results. The other subset or the total set of structures produce ensemble-averaged CD signals that disagree with the experimental results. These findings demonstrate the importance of CD constraints in the refinement of NMR derived structures of small proteins and peptides and 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 computing CD signals because of its strong and detailed CD spectrum and known solution structure.