Chimeric HTH motifs based on EF-hands

Kim, Youngbae; Welch, Joel T.; Lindstrom, Katherine M.; Franklin, Sonya J.

doi:10.1007/s007750000188

Cited by 34 publications

(67 citation statements)

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“…The design of peptide P3 has been described in greater detail elsewhere, and P3W was designed in an analogous manner (12). Briefly, crystal coordinates of calmodulin (1OSA) (19) and the engrailed homeodomain (1ENH) (20) were obtained from the Protein Data Bank and visualized by using the freeware program SWISS PDB VIEWER.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Briefly, crystal coordinates of calmodulin (1OSA) (19) and the engrailed homeodomain (1ENH) (20) were obtained from the Protein Data Bank and visualized by using the freeware program SWISS PDB VIEWER. The proteins were docked manually to overlay the HTH and the EF hand supersecondary structures as described (12). Peptides were synthesized by using standard Fmoc chemistry, cleaved from resin, and purified by HPLC to Ͼ95% purity [P3W, New England Peptide (Fitchburg, MA) (12).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…The proteins were docked manually to overlay the HTH and the EF hand supersecondary structures as described (12). Peptides were synthesized by using standard Fmoc chemistry, cleaved from resin, and purified by HPLC to Ͼ95% purity [P3W, New England Peptide (Fitchburg, MA) (12).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Alternately, native metal-binding sequences may be used in new contexts (12)(13)(14), and we have recently reported the de novo design (or more correctly, redesign) of metallopeptide nucleases based on this premise (12,15).…”

mentioning

confidence: 99%

“…The structural similarity in their natural protein contexts of the helix-turn-helix (HTH), a DNA-binding motif common to transcription factors, and the EF hand, a prevalent Ca-binding motif, led us to consider redesigning the DNA-binding turn to incorporate a metal site (12). These two supersecondary structures can both be classified as ␣Ϫ␣ corners, a ubiquitous protein structural element comprising two orthogonal ␣-helices tethered through a turn (16).…”

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confidence: 99%

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Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

Welch

Kearney

Franklin

2003

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Experimental Methodsmentioning

confidence: 99%