SPR設(shè)備在大分子復(fù)合物活化位點(diǎn)結(jié)合反應(yīng)分析中的應(yīng)用

ACS Chem. Biol. 2013, 8, 2794−2801  Michael P. Storz, ect.

美國(guó)化學(xué)生物學(xué)，2013.8.2794-2801  Michael P. Storz等人

Biochemical and Biophysical Analysis of a Chiral PqsD Inhibitor Revealing Tight-binding Behavior and Enantiomers with Contrary Thermodynamic Signatures

對(duì)PqsD抑制劑手性異構(gòu)體的生物化學(xué)和生物物理學(xué)分析揭示了其穩(wěn)定結(jié)合反應(yīng)和與其具有明顯相反熱動(dòng)力學(xué)特性的旋光異構(gòu)體的存在

Binding Site Analysis by SPR. To investigate whether our compounds bind in the active site, we performed binding experiments using SPR.

SPR分析結(jié)合位點(diǎn) 為了探究被檢復(fù)合物是否在活化位點(diǎn)結(jié)合，進(jìn)行了利用SPR技術(shù)的結(jié)合實(shí)驗(yàn)。

Surface Plasmon Resonance (SPR) Spectrometry. SPR binding studies were performed using a Reichert SR7500DC instrument optical biosensor (Reichert Technologies)。

表面等離子共振光譜儀 SPR結(jié)合實(shí)驗(yàn)使用了Reichert SR7500DC 光學(xué)傳感儀（Reichert Technologies，新型號(hào)為 2SPR）

圖一

Figure 1. Elucidation of the binding site by SPR. Addition of inhibitor to native PqsD leads to response curves (A) and (B) for compounds 2 and 3, respectively. In contrast to compound 2 (C), no response curve for 3 (D) is observed after addition to PqsD pretreated with ACoA.

圖1 利用SPR技術(shù)闡明結(jié)合位點(diǎn)。給天然PqsD加上抑制因子分別引起了A圖和B圖的信號(hào)曲線，分別對(duì)應(yīng)復(fù)合物2和3。相對(duì)于復(fù)合物2對(duì)應(yīng)的曲線（如圖C）,當(dāng)抑制因子加到被ACoA預(yù)處理過(guò)的PqsD時(shí)沒(méi)有出現(xiàn)復(fù)合物3應(yīng)有的反應(yīng)信號(hào)曲線（如圖D）。

Figure S9: Binding site analysis of (R)-3 and (S)-3 by SPR

圖S9：通過(guò)SPR技術(shù)對(duì)(R)-3和 (S)-3結(jié)合位點(diǎn)分析。

Figure S9. SPR experiment using 250 and 125μM of (R)-3 (blue) or (S)-3 (orange), respectively. Compounds were added to native PqsD and response curves (A) and (B) were recorded. No response was observed when identical concentrations of (R)-3 (C) or (S)-3 (D) were added to PqsD pretreated with ACoA. Furthermore, when (R)-3 was added until saturation of the SPR signal, subsequent addition of (S)-3 did not affect the observed response (E). The results indicate that the binding sites of both enantiomers are blocked by anthranilate.

圖S9，SPR實(shí)驗(yàn)中分別使用了250和125μM的(R)-3 (藍(lán)) or (S)-3 (橘)。復(fù)合物加入到自然PqsD中，并且記錄下了對(duì)應(yīng)的信號(hào)曲線(圖A) 和 (圖B)，當(dāng)同樣濃度(R)-3 (如圖C) 和 (S)-3 (如圖D)加到被ACoA預(yù)處理過(guò)的PqsD時(shí)沒(méi)有出現(xiàn)反應(yīng)信號(hào)（如圖D）。另外，當(dāng)持續(xù)加入(R)-3直到SPR信號(hào)飽和，隨后再加入(S)-3也不能影響反應(yīng)了（如圖E）。結(jié)果表明兩種異構(gòu)體的結(jié)合位點(diǎn)是被鄰氨基苯甲酸鹽封閉了。