The Design of Functional DNA-Binding Proteins Based on Zinc Finger Domains

Abstract

pus oocytes contained 7-11 zinc ions per protein, 21 led to the proposal that each 30-amino acid sequence bound one zinc ion through the conserved cysteine and histidine residues. This hypothesis was supported by limited proteolysis studies that yielded fragments differing in length by about 3 kDa, 21 suggesting that these proposed metal-binding units form individually folded, structurally stable domains.The nature of the metal binding sites in TFIIIA was directly probed by X-ray absorption spectroscopic methods. EXAFS analysis of the TFIIIA-5S RNA complex isolated from immature Xenopus oocytes was consistent with the proposed coordination with two sulfur atoms at 2.30 Å and two nitrogen atoms at 2.00 Å. 23 These distances matched reasonably closely those observed for zinc model complexes prepared by Koch and co-workers. 24,25 Zinc Finger Domain Peptides with Naturally Occurring SequencesThe zinc finger hypothesis suggested a reductionist approach to the characterization of these domains. Derek Jantz was born in Littleton, Colorado, and graduated from the University of Colorado at Boulder (B.A. 1996). He received his Ph.D. from the Johns Hopkins University School of Medicine in 2003 under the direction of Jeremy Berg. His research concerned the DNA-binding characteristics of designed Cys 2 His 2 zinc finger proteins. He is presently working as a Postdoctoral Fellow with Homme Hellinga at Duke University Medical Center, focusing on computational protein design. He was selected as graduate student speaker at his graduation from Johns Hopkins, where he described graduate school as "the best period of indeterminate length of my life". Barbara T. Amann was born and raised in Swarthmore, Pennsylvania. She received her B.A. in chemistry from Mt. Holyoke College in 1983. She developed an interest in bioinorganic chemistry at the Pennsylvania State University, where she received her Ph.D. working with Bill Horrocks on lanthanide substitution of the calcium-binding protein calmodulin. In 1988, she joined Jeremy Berg's laboratory as a Postdoctoral Fellow in the Chemistry Department of Johns Hopkins University. She moved with the Berg laboratory to the Department of Biophysics and Biophysical Chemistry at Johns Hopkins School of Medicine and became a Research Associate. In this position, she has studied a range of zinc-binding domains, primarily by NMR, and has run the NMR facility of the department. She has enjoyed teaching other researchers NMR methods and assisting them with their projects. She loves spending time with her husband, two children, dog, and snake. Gregory J. Gatto, Jr., was raised in East Brunswick, New Jersey. He received his A.B. in chemistry in 1994 from Princeton University, where he worked in the laboratory of Martin F. Semmelhack. In the spring of 2003, he received his M.D. and Ph.D. degrees from Johns Hopkins University School of Medicine. There, he worked in the laboratory of Jeremy Berg on the structural biology of designed zinc finger proteins and the molecular details of peroxisomal targeting sig...

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

“…Z inc-finger proteins (ZFs) are a large class of proteins that use zinc as structural cofactors (1)(2)(3)(4). ZFs perform a variety of functions ranging from the modulation of gene expression through specific interactions with DNA or RNA to the control of signaling pathways via protein-protein interactions.…”

“…The general feature that defines a ZF protein is the presence of one or more domains that contain a combination of four cysteine and/or histidine residues that serve as ligands for zinc. When zinc binds to these ligands, the domain adopts the structure necessary for function (1)(2)(3)(4).…”

“…This class is composed of ZFs that contain a Cys 2 His 2 domain (CysX 2-5 CysX 12-13 HisX 3-5 His). Classical ZFs adopt an alphahelical/antiparallel beta-sheet structure when zinc is coordinated and bind DNA in a sequence-specific manner (2,4). The remaining classes of ZFs are collectively called "nonclassical" ZFs (1).…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…It is structurally resembling to the well-known zinc-finger motif (also possessing a ββα-metal binding structure [92]) but its function is different. The classical active site of the HNH nucleases is characterized by a HX 10-17 NX 6-11 (H/N) motif, where X stands for any amino acid with the exception of those with side-chains for strong metal ion binding or proline.…”

Targeting Nucleic Acids using Dynamic Combinatorial Chemistry

“…[1][2][3] The flexibility of these biomolecules leads to a diversity of secondary structures, whose formation and stability is highly dependent on the base sequence and environment. [4,5] The study of small molecules that recognize and bind with high affinity and selectivity to duplex, triplex, and quadruplex DNA, as well as hairpins, bulges, and RNA loops has attracted significant interest because of the involvement of many of these structures in disease.…”