A minimalist Cys2His2 zinc finger peptide, Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-LysAla-Ala-Leu-Ala-Ala-His-Ala-Ala-Ala-His-Ala-Lys, has been synthesized. Metal binding studies using Co2+ as a probe indicated that this peptide forms a 1:1 peptide/metal complex with a dissociation constant comparable to that observed for other zinc finger peptides. At high peptide concentrations, a 2:1 peptide/metal complex also forms, with four cysteinates coordinated to Co2+. Additional studies with sequence variants in which the canonical hydrophobic residues were changed to alanine, or in which one of the residues between the cysteines and the histidines was deleted, revealed an even more pronounced formation of the 2:1 complex over the 1:1 complex. In addition, the absorption spectra of the 1:1 peptide/Co2+ complexes of the variant peptides are significantly different from those seen for complexes of the parent peptide or those of more typical zinc finger peptides. NMR studies revealed that the parent peptide folds in the presence of Zn2+ to a structure very similar to that observed for other zinc finger peptides of this class. Taken together, these results suggest that the metalbinding and canonical hydrophobic residues are necessary and sufficient to determine the structure of this class of zinc finger peptides.The amino acid sequence of a protein contains the information sufficient to determine its three-dimensional structure (1). A variety of experiments have demonstrated that this information is not distributed uniformly but that a smaller set of residues are crucial for correct structure formation. For example, the elegant work of Kaiser and Kezdy (2) showed that simplified (or minimalist) versions of naturally occurring peptides could be designed and synthesized, retaining many of the structural (and, in many cases, functional) properties of the native materials. This approach has been extended to more complex oligomeric structures by DeGrado et al. (3). The uneven distribution of folding information has also been demonstrated by Sauer and coworkers (4) using combinatorial mutagenesis. Here, a protein sequence is randomized in a number of positions, functional molecules are selected, and their amino acid sequences are determined. It has been found that some positions are quite tolerant ofa wide range ofamino acids whereas others have very stringent restrictions. These observations suggest that a large number of sequences that have only a small subset of residues fixed may fold in functionally equivalent manners. The Cys2His2 zinc finger sequences typified by transcription factor TFIIIA may represent a naturally occurring set of sequences of this type. After the determination of the sequence of a cDNA clone of TFIIIA (5), it was noted that the deduced amino acid sequence contained nine tandem sequences that shared the consensus (Phe,Tyr)-Xaa-Cys-Xaa2,4-Cys-Xaa3-Phe-Xaa5-Leu-Xaa2-His-Xaa3,4-His-Xaa2-6 where Xaa represents relatively variable amino acids (6,7). Subsequently, a large number of other ...
On the basis of computer simulation a mechanism for photosensitized thymine dimerization involving association between thymine and photosensitizer is proposed.
Since thymine dimerization is the main photochemical lesion occuring in uv irradiated DNA, an understanding of the mechanism of dimerization is biologically significant. Both photosensitized and direct dimerization are important in DNA, but because photosensitized thymine dimerization has been less thoroughly investigated, this has been the major topic of study in this laboratory. By comparing experimental results with those obtained by computer simulation, attempts have been made to deduce mechanisms for photosensitization by acetone, acetophenone, and benzophenone.Photolysis of photosensitized solutions was performed using a xenon lamp and quantitative detection of dimer was achieved using h.p.1.c. techniques. A program designed to solve differential rate equations was used for the computer simulation of reaction mechanisms.Based on the results obtained it has been confirmed that acetone photosensitization over the entire range of thymine concentration considered mol dm ' to 10.' mol dm ') proceeds via diffusion controlled triplet transfer from the photosensitizer to thymine, followed by bimolecular collision of ground and excited state thymine monomers. For acetophenone and benzophenone photosensitization this method applied a t low thymine concentrations, but a t higher concentrations (
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