A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.
A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in ␣-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ؎ 0.3 M) and for Eu(III) (6.2 ؎ 0.3 M) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helixturn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metalbinding loop but also retains the ␣؊␣ corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence.
One of the great promises of de novo protein design is the possibility of incorporating new reactivity or selectivity into a protein matrix for specific chemical targets or to advance biological understanding. The redesign of a known protein fold is one powerful approach to achieving new function, and the inclusion of metal sites that could confer a range of enzymatic activities is particularly intriguing. Designed metalloproteins have the potential to promote natural enzymatic reactions on unnatural substrates or even to achieve reactivity through mechanisms completely unprecedented in biology. However, to interpret and evaluate their activity and to improve subsequent designs, the resultant constructs must retain a predictable well-folded structure. A structurally well characterized robust scaffold is therefore fundamental to the design of minimalist enzymes.In recent years, several approaches have been taken to engineer novel metalloproteins, including building a metal site and scaffold completely from first principles (1-5), grafting ligands onto known protein scaffolds by point mutations that were identified through modeling or a vector-based algorithm such as DENZYMER (5-9), or redesigning natural metalloproteins for potential new reactivity (10, 11). Alternately, native metal-binding sequences may be used in new contexts (12-14), and we have recently reported the de novo design (or more correctly, redesign) of metallopeptide nucleases based on this premise (12, 15).The structural similarity in their natural protein contexts of the helix-turn-helix (HTH), a DNA-binding motif c...
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