生物谷報(bào)道：在4月30號(hào)出版的《美國(guó)化學(xué)會(huì)志》（JACS）上報(bào)道了武漢物數(shù)所楊俊博士在美國(guó)特拉華大學(xué)用固體NMR新方法研究蛋白質(zhì)界面的研究工作,。
一些生物大分子,，如膜蛋白，蛋白質(zhì)復(fù)合體,，蛋白質(zhì)纖維等,，在生命過(guò)程中起著極為重要的作用，但是由于難以得到這些生物分子的單晶以及它們?cè)谌芤褐械牡腿芙舛龋?用X-ray和液體NMR很難得到它們的結(jié)構(gòu),。一個(gè)典型的例子是膜蛋白質(zhì),。膜蛋白約占與人類基因編碼有關(guān)的蛋白質(zhì)的30%，一些重要的生命活動(dòng)如能量轉(zhuǎn)換,、信息識(shí)別與傳遞,、物質(zhì)運(yùn)送和分配都與膜蛋白密切相關(guān)。但是到目前為止,，只有157種（總共約3萬(wàn)種）膜蛋白的三維結(jié)構(gòu)是已知的,。對(duì)于這些“困難”的生物大分子，固體NMR被認(rèn)為是最有前途的研究手段之一,。自從2002年德國(guó)科學(xué)家首次用魔角旋轉(zhuǎn)NMR得到固體蛋白質(zhì)的三維結(jié)構(gòu)以來(lái),，這幾年這個(gè)領(lǐng)域飛速向前發(fā)展,。隨著高磁場(chǎng)NMR儀器的使用，魔角旋轉(zhuǎn)NMR探頭技術(shù)的發(fā)展,，固體蛋白質(zhì)樣品制備技術(shù)的成熟和一批兩維到四維固體NMR脈沖序列的使用,，魔角旋轉(zhuǎn)NMR研究蛋白質(zhì)的能力大大提高，魔角旋轉(zhuǎn)NMR已經(jīng)能夠?qū)?5-30 KDa的蛋白質(zhì)進(jìn)行NMR信號(hào)全歸屬和相應(yīng)的結(jié)構(gòu)和動(dòng)力學(xué)研究,。
在這個(gè)研究中,，楊俊和特拉華大學(xué)的同事Tatyana Polenova設(shè)計(jì)了一組新脈沖序列，他們用這組脈沖序列研究了用不同同位素標(biāo)記的thioredoxin蛋白質(zhì)組裝體的分子內(nèi)和分子間的界面,。首先他們用理論模擬和NMR實(shí)驗(yàn)證實(shí)了固體NMR中的REDOR技術(shù)可以用來(lái)消除13C,15N全富集的蛋白質(zhì)主鏈上的15N信號(hào),，實(shí)現(xiàn)了用一個(gè)蛋白質(zhì)樣品同時(shí)進(jìn)行NMR信號(hào)歸屬和蛋白質(zhì)界面研究。借助于對(duì)遠(yuǎn)程相互作用敏感的1H/13C REDOR和PAIN－CP技術(shù), 他們?cè)O(shè)計(jì)了兩個(gè)脈沖序列,，用不同核自旋對(duì)的相關(guān)性觀察到了蛋白質(zhì)界面上空間相近的殘基對(duì),。另外，他們還設(shè)計(jì)了兩個(gè)脈沖序列對(duì)蛋白質(zhì)另外一段的主鏈上的15N信號(hào)進(jìn)行了歸屬,。這組固體NMR的脈沖序列和相應(yīng)的同位素標(biāo)記方法將可以在更大的蛋白質(zhì)復(fù)合體的界面研究中使用,。（來(lái)源：中國(guó)科學(xué)院武漢物理與數(shù)學(xué)研究所）
生物谷推薦原始出處：
JACS，130 (17), 5798–5807,，Jun Yang,，Tatyana Polenova
Magic Angle Spinning NMR Experiments for Structural Studies of Differentially Enriched Protein Interfaces and Protein Assemblies
Jun Yang,† Maria Luisa Tasayco,‡ and Tatyana Polenova*†
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, and Department of Chemistry, The City College of New York, Convent Avenue at 138th Street, New York, New York 10031
Abstract:
Protein–protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1–73(13C,15N)/74–108(15N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range 15N−13C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the 15N proton-driven spin diffusion and into the 1H−15N FSLG−HETCOR sequences permits 1H and 15N resonance assignments of the 74–108(15N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.
