Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences.

Peptides that mediate dimerization of attached zinc finger DNA-binding domains have been evolved in vitro starting from random sequences. We first used phage display to select dimerization elements from libraries of random 15-residue polypeptides that were fused to the N terminus of the zinc finger domains. We then reoptimized these peptides by sequentially randomizing five-residue blocks (proceeding across the peptide in three steps) and selecting variant peptides that further stabilized the protein-DNA complex. Biochemical experiments confirmed that the selected peptides promote dimerization of the zinc fingers on an appropriate DNA target site. These results demonstrate that dimerization units can be obtained readily from random polypeptide libraries of moderate complexity. Our success reemphasizes the utility of searching random peptide libraries in protein design projects, and the sequences presented here may be useful when designing novel transcription factors.

[1]  D Eisenberg,et al.  3D domain swapping: A mechanism for oligomer assembly , 1995, Protein science : a publication of the Protein Society.

[2]  T. Laue.  Sedimentation equilibrium as thermodynamic tool. , 1995, Methods in enzymology.

[3]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[4]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[5]  Enrico A. Stura,et al.  Functional Mimicry of a Protein Hormone by a Peptide Agonist: The EPO Receptor Complex at 2.8 Å , 1996, Science.

[6]  C. Pabo,et al.  Structure-based design of a dimeric zinc finger protein. , 1998, Biochemistry.

[7]  Donald F. Senear,et al.  [9] Quantitative DNase footprint titration: A method for studying protein-DNA interactions , 1986 .

[8]  M L Johnson,et al.  Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. , 1981, Biophysical journal.

[9]  Ronald W. Barrett,et al.  Small Peptides as Potent Mimetics of the Protein Hormone Erythropoietin , 1996, Science.

[10]  B. Rost,et al.  Prediction of protein secondary structure at better than 70% accuracy. , 1993, Journal of molecular biology.

[11]  H. Lowman,et al.  Bacteriophage display and discovery of peptide leads for drug development. , 1997, Annual review of biophysics and biomolecular structure.

[12]  James C. Hu,et al.  Genetic selection of short peptides that support protein oligomerization in vivo , 1999, Current Biology.

[13]  C. Wolberger,et al.  Crystal Structure of the MATa1/MATα2 Homeodomain Heterodimer Bound to DNA , 1995, Science.

[14]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[15]  Identification of Disease Genes in Genetic Isolates , 1996, Methods.

[16]  N. Pavletich,et al.  Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A , 1991, Science.

[17]  J. Wells,et al.  Hormone phage: An enrichment method for variant proteins with altered binding properties , 1990, Proteins.

[18]  C. Pabo,et al.  Phage display methods for selecting zinc finger proteins with novel DNA-binding specificities. , 1996, Methods in Enzymology.

[19]  C. Pabo,et al.  Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. , 1996, Genes & development.

[20]  Aneel K. Aggarwal,et al.  Structure of a DNA-bound Ultrabithorax–Extradenticle homeodomain complex , 1999, Nature.

[21]  L. Lau,et al.  A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. S. Kim,et al.  A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. , 1993, Science.

[23]  J. Carey.  [8] Gel retardation , 1991 .