5 research outputs found
A Set of <i>de Novo</i> Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub‑nanomolar Regime
The availability of peptide and protein
components that fold to
defined structures with tailored stabilities would facilitate rational
protein engineering and synthetic biology. We have begun to generate
a toolkit of such components based on <i>de novo</i> designed
coiled-coil peptides that mediate protein–protein interactions.
Here, we present a set of coiled-coil heterodimers to add to the toolkit.
The lengths of the coiled-coil regions are 21, 24, or 28 residues,
which deliver dissociation constants in the micromolar to sub-nanomolar
range. In addition, comparison of two related series of peptides highlights
the need for including polar residues within the hydrophobic interfaces,
both to specify the dimer state over alternatives and to fine-tune
the dissociation constants
<i>De Novo</i>-Designed α‑Helical Barrels as Receptors for Small Molecules
We
describe <i>de novo</i>-designed α-helical barrels
(αHBs) that bind and discriminate between lipophilic biologically
active molecules. αHBs have five or more α-helices arranged
around central hydrophobic channels the diameters of which scale with
oligomer state. We show that pentameric, hexameric, and heptameric
αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene
(DPH) in the micromolar range and fluoresce. Displacement of the dye
is used to report the binding of nonfluorescent molecules: palmitic
acid and retinol bind to all three αHBs with submicromolar inhibitor
constants; farnesol binds the hexamer and heptamer; but β-carotene
binds only the heptamer. A co-crystal structure of the hexamer with
farnesol reveals oriented binding in the center of the hydrophobic
channel. Charged side chains engineered into the lumen of the heptamer
facilitate binding of polar ligands: a glutamate variant binds a cationic
variant of DPH, and introducing lysine allows binding of the biosynthetically
important farnesol diphosphate
<i>De Novo</i>-Designed α‑Helical Barrels as Receptors for Small Molecules
We
describe <i>de novo</i>-designed α-helical barrels
(αHBs) that bind and discriminate between lipophilic biologically
active molecules. αHBs have five or more α-helices arranged
around central hydrophobic channels the diameters of which scale with
oligomer state. We show that pentameric, hexameric, and heptameric
αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene
(DPH) in the micromolar range and fluoresce. Displacement of the dye
is used to report the binding of nonfluorescent molecules: palmitic
acid and retinol bind to all three αHBs with submicromolar inhibitor
constants; farnesol binds the hexamer and heptamer; but β-carotene
binds only the heptamer. A co-crystal structure of the hexamer with
farnesol reveals oriented binding in the center of the hydrophobic
channel. Charged side chains engineered into the lumen of the heptamer
facilitate binding of polar ligands: a glutamate variant binds a cationic
variant of DPH, and introducing lysine allows binding of the biosynthetically
important farnesol diphosphate
<i>De Novo</i>-Designed α‑Helical Barrels as Receptors for Small Molecules
We
describe <i>de novo</i>-designed α-helical barrels
(αHBs) that bind and discriminate between lipophilic biologically
active molecules. αHBs have five or more α-helices arranged
around central hydrophobic channels the diameters of which scale with
oligomer state. We show that pentameric, hexameric, and heptameric
αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene
(DPH) in the micromolar range and fluoresce. Displacement of the dye
is used to report the binding of nonfluorescent molecules: palmitic
acid and retinol bind to all three αHBs with submicromolar inhibitor
constants; farnesol binds the hexamer and heptamer; but β-carotene
binds only the heptamer. A co-crystal structure of the hexamer with
farnesol reveals oriented binding in the center of the hydrophobic
channel. Charged side chains engineered into the lumen of the heptamer
facilitate binding of polar ligands: a glutamate variant binds a cationic
variant of DPH, and introducing lysine allows binding of the biosynthetically
important farnesol diphosphate
Design and Nuclear Magnetic Resonance (NMR) Structure Determination of the Second Extracellular Immunoglobulin Tyrosine Kinase A (TrkAIg2) Domain Construct for Binding Site Elucidation in Drug Discovery
The tyrosine kinase A (TrkA) receptor
is a validated therapeutic
intervention point for a wide range of conditions. TrkA activation
by nerve growth factor (NGF) binding the second extracellular immunoglobulin
(TrkAIg2) domain triggers intracellular signaling cascades. In the
periphery, this promotes the pain phenotype and, in the brain, cell
survival or differentiation. Reproducible structural information and
detailed validation of protein–ligand interactions aid drug
discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped
dimer in the absence of NGF, occluding the binding surface. Here we
report the design and structural validation by nuclear magnetic resonance
spectroscopy of the first stable, biologically active construct of
the TrkAIg2 domain for binding site confirmation. Our structure closely
mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the <sup>1</sup>H–<sup>15</sup>N correlation spectra confirm that both
NGF and a competing small molecule interact at the known binding interface
in solution