We report the 470-MHz (11.7 T) 19F solution nuclear magnetic resonance (NMR) spectra of 2-, 3-, and 4-fluorophenylalanine incorporated into the egg white lysozymes (EC 3.2.1.17) of chicken, pheasant, and duck, as well as spectra of 4-fluorotryptophan incorporated into chicken, California valley quail, and Bob White quail and 5- and 6-fluorotryptophan-labeled chicken lysozyme. The 19F solution NMR spectrum of [4-F]Phe hen egg white lysozyme (HEWL) consists of three sharp resonances, which span a total chemical shift range of 4.8 ppm (at p2H = 6.1). For [3-F]Phe HEWL, the chemical shift range is much smaller, 1.0 ppm (at p2H = 5.9), due presumably to the occurrence of fast phenyl ring flips about the C beta-C gamma bond axis. For [2-F]Phe HEWL, six resonances are observed, spanning a chemical shift range of 7.4 ppm (at p2H = 5.8), due to slow C beta-C gamma ring flips, i.e., both ring-flip isomers appear to be "frozen in" because of steric hindrance. Rotation of the [2-F]Phe residues remains slow up to 55 degrees C (p2H = 4.7). With the [F]Trp-labeled proteins, we find a maximal 14.6-ppm shielding range for [4-F]Trp HEWL but only a 2.8- and 2.4-ppm range for [5- and 6-F]Trp HEWL, respectively, due presumably to increased solvent exposure in the latter cases. Guanidinium chloride denaturation causes loss of essentially all chemical shift nonequivalence, as does thermal denaturation. Spectra recorded as a function of pH show relatively small chemical shift changes (< 1.4 ppm) over the pH range of approximately 1.2-7.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Duocarmycin A (Duo) normally alkylates adenine N3 at the 3 end of A؉T-rich sequences in DNA. The efficient adenine alkylation by Duo is achieved by its monomeric binding to the DNA minor groove. The addition of another minor groove binder, distamycin A (Dist), dramatically modulates the site of DNA alkylation by Duo, and the alkylation switches preferentially to G residues in G؉C-rich sequences. HPLC product analysis using oligonucleotides revealed a highly efficient G-N3 alkylation via the cooperative binding of a heterodimer between Duo and Dist to the minor groove. The three-dimensional structure of the ternary alkylated complex of Duo͞Dist͞d(CAGGTGGT)⅐d(ACCACCTG) has been determined by nuclear Overhauser effect (NOE)-restrained refinement using 750 MHz two-dimensional NOE spectroscopy data. The refined NMR structure fully explains the sequence requirement of such modulated alkylations. This is the first demonstration of Duo DNA alkylation through cooperative binding with another structurally different natural product, and it suggests a promising new way to alter or modify the DNA alkylation selectivity in a predictable manner.

The anticancer drug actinomycin D (ActD) binds to DNA by intercalating its phenoxazone ring at a GpC step with the drug's two cyclic pentapeptides located in the DNA minor groove. The binding affinity to the GpC site is influenced by the flanking sequences. We have analyzed the structure of the complexes of ActD−d(GAAGCTTC)2 and ActD−d(GATGCTTC)2 by NOE-restrained refinement. Binding of ActD to the −(AGCT)2− sequence causes the N-methyl group of MeVal to wedge between the bases at the ApG step, resulting in kinks on both sides of the intercalator site. Surprisingly ActD forms a very stable complex with d(GATGCTTC)2 in which the same methyl group now fits snugly in a cavity at the TpG step created by the T:T mismatched base pair. In contrast, ActD does not stabilize the unstable A:A−mismatched d(GAAGCATC)2 duplex to a significant extent. Such high-resolution structural information helps reveal the sequence preference of ActD toward −XGCY− tetranucleotides. The triplet repeat (CAG) n and (CTG) n motifs, which are associated with several genetic diseases such as Huntington's disease/spinobulbar muscular atrophy and myotonic dystrophy, contain −AGCA− and −TGCT− sequences. It was found by NMR spectroscopic studies that ActD significantly stabilizes the mismatched (CAG) n and (CTG) n duplexes and prevents them from annealing with each other to form the Watson−Crick duplex. This suggests that ActD may trap the cruciform structure of the (CAG) n /(CTG) n sequence and may exert certain biological actions (e.g., stopping the expansion during replication), since interference of the equilibrium between the duplex and cruciform structures by proteins or drugs may have biological consequences.

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