Circular Dichroism Constrains NMR-Derived Structures of a Folded Trinitrophenylated Hexapeptide in Solution Thomas P. Burghardt,* ,† Nenad Juranic ´ , † Slobodan Macura, † Andras Muhlrad, ‡ and Katalin Ajtai † Contribution from the Department of Biochemistry, Mayo Foundation, Rochester, MN, and Department of Oral Biology, Hebrew UniVersity, Jerusalem, Israel ReceiVed May 3, 1999. ReVised Manuscript ReceiVed August 19, 1999 Abstract: A trinitrophenylated hexapeptide, with sequence imitating that near the reactive lysyl residue (Lys 84 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 1 H 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. Introduction A small peptide of known structure is a useful construct for studying protein characteristics related to the elementary determiners of conformation or folding pathway. A significant problem for solving small peptide structures with proton nuclear magnetic resonance ( 1 H NMR) is ambiguity in assigning correct chirality to all or a part of the molecule since handedness is not constrained by the atomic distances. In addition, there is a question of the uniqueness of the NMR-derived structures because small peptides may exist as a large ensemble of con- formers. The chiral ambiguity in the NMR structures might be removed, and the number of conformers in the ensemble re- duced, by comparison of the peptide circular dichroism (CD) to a theoretical signal prediction computed from trial peptide structures. Essential for successful combination of CD with NMR data is a reliable CD signal calculation method applicable to peptides under experimental conditions. The near-ultraviolet (UV) CD spectrum is sensitive to secon- dary and tertiary structure of a polypeptide. Characteristics of this signal are calculable from the nature of the electromagnetic interactions using the polypeptide atomic structure. 1,2 Known approximate and tractable methods produce calculated near UV CD spectra from protein crystallographic coordinates that agree with spectra observed from the protein in solution. 3,4 Assump- tions implicit in these calculations are that (i) crystal coordinates provide the predominant solution structure and that (ii) interac- tions contributing significantly to the signal are from residues near the signal source (e.g., all residues within a certain radius of a tryptophan). These assumptions are relaxed when consider- ing a small polypeptide that folds into a well-defined structure in solution and contains a suitable signal donor. Then 1 H NMR can characterize the peptide’s solution structure, notwithstanding the chiral ambiguity, and the spectroscopic signal calculation can include interactions from all residues. A peptide suitable for this application has six residues taken from the chicken skeletal myosin sequence surrounding the reactive lysine residue (RLR or Lys 84 ). 5 The hexapeptide (6p) is trinitrophenylated at the side chain ǫ-amino group of Lys 3 , the lysine residue corresponding to RLR. The trinitrophenyl (TNP) group in the hexapeptide (TNP-6p), acting as the signal donor in our calculation, has an observable induced CD signal in the three lowest-energy transitions of its absorption band. This folding peptide provides the means to test our implementa- tion of the methods for calculating the near-UV CD spectrum of a peptide and to utilize suggestions from the CD to refine the 1 H NMR structure determination. Materials and Methods Chemicals. Dimethyl sulfoxide (DMSO) and HEPES were from Sigma (St. Louis, MO). The 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Fluka (Milwaukee, WI), DMSO-d6 was from Isotec Inc. * Corresponding author. † Mayo Foundation. ‡ Hebrew University. (1) Tinoco, I. J. Chem. Phys. 1960, 33, 1332-1338. (2) Bayley, P. M.; Nielsen, E. B.; Schellman, J. A. J. Phys. Chem. 1969, 73, 228-243. (3) Strickland, E. H. Biochemistry 1972, 11, 3465-3474. (4) Goux, W. J.; Hooker, T. M. J. Am. Chem. Soc. 1980, 102, 7080- 7087. (5) Ajtai, K.; Peyser, Y. M.; Park, S.; Burghardt, T. P.; Muhlrad, A. Biochemistry 1999, 38, 6428-6440. 10373 J. Am. Chem. Soc. 1999, 121, 10373-10378 10.1021/ja991445u CCC: $18.00 © 1999 American Chemical Society Published on Web 10/21/1999