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Two Engineered OBPs with opposite temperature-dependent affinities towards 1-aminoanthracene.

ABSTRACT: Engineered odorant-binding proteins (OBPs) display tunable binding affinities triggered by temperature alterations. We designed and produced two engineered proteins based on OBP-I sequence: truncated OBP (tOBP) and OBP::GQ20::SP-DS3. The binding affinity of 1-aminoanthracene (1-AMA) to these proteins revealed that tOBP presents higher affinity at 25?°C (kd?=?0.45??M) than at 37?°C (kd?=?1.72??M). OBP::GQ20::SP-DS3 showed an opposite behavior, revealing higher affinity at 37?°C (kd?=?0.58??M) than at 25?°C (kd?=?1.17??M). We set-up a system containing both proteins to evaluate their temperature-dependent binding. Our data proved the 1-AMA differential and reversible affinity towards OBPs, triggered by temperature changes. The variations of the binding pocket size with temperature, confirmed by molecular modelling studies, were determinant for the differential binding of the engineered OBPs. Herein we described for the first time a competitive temperature-dependent mechanism for this class of proteins.

SUBMITTER: Goncalves F

PROVIDER: S-EPMC6172251 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Two Engineered OBPs with opposite temperature-dependent affinities towards 1-aminoanthracene.

Gonçalves Filipa F Castro Tarsila G TG Azoia Nuno G NG Ribeiro Artur A Silva Carla C Cavaco-Paulo Artur A

Scientific reports 20181004 1

Engineered odorant-binding proteins (OBPs) display tunable binding affinities triggered by temperature alterations. We designed and produced two engineered proteins based on OBP-I sequence: truncated OBP (tOBP) and OBP::GQ<sub>20</sub>::SP-DS3. The binding affinity of 1-aminoanthracene (1-AMA) to these proteins revealed that tOBP presents higher affinity at 25 °C (kd = 0.45 μM) than at 37 °C (kd = 1.72 μM). OBP::GQ<sub>20</sub>::SP-DS3 showed an opposite behavior, revealing higher affinity at 37 ...[more]

PMID: 30287882

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Project description:The alpha(v)beta(3) integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to alpha(v)beta(3) integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized alpha(v)beta(3) integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing alpha(v)beta(3) integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to alpha(v)beta(3) integrins and had only minimal or no binding to alpha(v)beta(5), alpha(5)beta(1), and alpha(iib)beta(3) integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for alpha(v)beta(3) and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against alpha(v)beta(3) integrins resulted in peptides that retained high affinities for alpha(v)beta(3) and had increased specificities for alpha(v)beta(3) over alpha(iib)beta(3) integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.

Dataset Information

Two Engineered OBPs with opposite temperature-dependent affinities towards 1-aminoanthracene.

Publications

Two Engineered OBPs with opposite temperature-dependent affinities towards 1-aminoanthracene.

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