為鑑定和蛋白質產生交互作用之配體所進行的化合物庫生物篩檢有其效率，但通常無法揭示配體會是否會對其功能特性產生干擾，或是如何干擾。For this a biochemical/functional assay is required.But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput.
在此我們探索了一款高通量二次諧波產生 (Second-Harmonic Generation，SHG) 生物感測器，此感測器可即時偵測與蛋白質結合時誘發構形變化的片段，並找出動態區域。Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs).They were conjugated with second harmonic-active labels via amine or maleimide coupling.
為鑑定和蛋白質產生交互作用之配體所進行的化合物庫生物篩檢有其效率，但通常無法揭示配體會是否會對其功能特性產生干擾，或是如何干擾。For this a biochemical/functional assay is required.But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput.
在此我們探索了一款高通量二次諧波產生 (Second-Harmonic Generation，SHG) 生物感測器，此感測器可即時偵測與蛋白質結合時誘發構形變化的片段，並找出動態區域。Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs).They were conjugated with second harmonic-active labels via amine or maleimide coupling.
To validate the assay, it was confirmed that the conformational changes induced in AChBP by nicotinic acetyl choline receptor (nAChR) agonists and antagonists were qualitatively different.A 1056 fragment library was subsequently screened against all variants and conformational modulators of AChBP were successfully identified, with hit rates from 9–22%, depending on the AChBP variant.A subset of four hits was selected for orthogonal validation and structural analysis.A time-resolved grating-coupled interferometry-based biosensor assay confirmed the interaction to be a reversible 1-step 1 : 1 interaction, and provided estimates of affinities and interaction kinetic rate constants (KD = 0.28–63 μM, ka = 0.1–6 μM−1 s−1, kd = 1 s−1).X-ray crystallography of two of the fragments confirmed their binding at a previously described conformationally dynamic site, corresponding to the regulatory site of LGICs.
These results reveal that SHG has the sensitivity to identify fragments that induce conformational changes in a protein.A selection of fragment hits with a response profile different to known LGIC regulators was characterized and confirmed to bind to dynamic regions of the protein.
