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Accessibility, reactivity, and selectivity of side chains within a channel of de Novo peptide assembly

DOI: 10.1021/ja4053027 DOI Help
PMID: 23924058 PMID Help

Authors: Antony Burton (University of Bristol) , Franziska Thomas (University of Bristol) , Christopher Agnew (University of Bristol) , Kieran L. Hudson (University of Bristol) , Stephen E. Halford (University of Bristol) , Leo Brady (University of Bristol) , Derek N. Woolfson (University of Bristol)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society , VOL 135 (34)

State: Published (Approved)
Published: August 2013

Abstract: Ab initio design of enzymes requires precise and predictable positioning of reactive functional groups within accessible and controlled environments of de novo protein scaffolds. Here we show that multiple thiol moieties can be placed within a central channel, with approximate dimensions 6 × 42 Å, of a de novo, six-helix peptide assembly (CC-Hex). Layers of six cysteine residues are introduced at two different sites ∼6 (the “L24C” mutant) and ∼17 Å (L17C) from the C-terminal opening of the channel. X-ray crystal structures confirm the mutant structures as hexamers with internal free thiol, rather than disulfide-linked cysteine residues. Both mutants are hexa-alkylated upon addition of iodoacetamide, demonstrating accessibility and full reactivity of the thiol groups. Comparison of the alkylation and unfolding rates of the hexamers indicates that access is directly through the channel and not via dissociation and unfolding of the assembly. Moreover, neither mutant reacts with iodoacetic acid, demonstrating selectivity of the largely hydrophobic channel. These studies show that it is possible to engineer reactive side chains with both precision and control into a de novo scaffold to produce protein-like structures with chemoselective reactivity.

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 14/08/2013 10:44

Discipline Tags:

Biochemistry Chemistry Structural biology Biophysics Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)