Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein
Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein
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DOI：
10.1006/abbi.2000.1726
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发表时间：
2000-04-15
影响因子：
3.9
通讯作者：
Markley, JL
中科院分区：
文献类型：
--
作者：
Assadi-Porter, FM;Aceti, DJ;Markley, JL
Brazzein最初是从非洲植物Pentadiplandra brazzeana Baillon的果实中分离出来的，是已知具有内在甜味的蛋白质中最小、最耐热和最稳定pH的成员。这些特性使Brazzein成为研究甜味蛋白质的化学和结构要求的理想系统。我们使用了蛋白质(J，E，Caldwell et at(1998)Nut，Struct，Biol)的三维结构。5,427-431)，作为设计15个合成基因表达结构的指南，目的是描绘巴西玉米素的甜度决定因素。在大肠杆菌中异源生产蛋白质，分离和纯化，如伴随论文(assadi-Porter，F.NI，，Aceti，D，，Cheng，H，和Markley，J，L，，本期)所述。一维(1)氢核磁共振波谱分析表明，在所使用的条件下，除了一个之外，所有这些变体都正确折叠。品尝小组比较了这些蛋白质溶液的味觉特性与从水果中分离的蔗糖和巴西玉米素的味道特性。在des-pGlu1-brazzein背景的14个突变中，有4个几乎没有甜度，6个显著降低了甜度，2个具有与des-pGlu1-brazzein相同的味道(甜度是从含有pGlu1的水果中分离出来的主要形式brazzein的两倍)，还有两个甜度大约是des-pGlu1-brazzein的两倍，总体而言，结果表明该蛋白质的两个区域对brazzein的甜度至关重要：一个区域包括蛋白质的N-和C-末端，这两个区域彼此靠近，另一个区域包括围绕Arg43的灵活环，(C)2000学术出版社。
Brazzein, originally isolated from the fruit of the African plant Pentadiplandra brazzeana Baillon, is the smallest, most heat-stable and pH-stable member of the set of proteins known to have intrinsic sweetness. These properties make brazzein an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. We have used the three-dimensional structure of the protein (J, E, Caldwell et at (1998) Nut, Struct, Biol. 5, 427-431) as a guide in designing 15 synthetic genes in expression constructs aimed at delineating the sweetness determinants of brazzein. Protein was produced heterologously in Escherichia coli, isolated, and purified as described in the companion paper (Assadi-Porter, F. NI,, Aceti, D,, Cheng, H,, and Markley, J, L,, this issue). Analysis by one-dimensional (1)H NMR spectroscopy indicated that all but one of these variants had folded properly under the conditions used, A taste panel compared the gustatory properties of solutions of these proteins to those of sucrose and brazzein isolated from fruit. Of the 14 mutations in the des-pGlu1-brazzein background, four exhibited almost no sweetness, six had significantly reduced sweetness, two had taste properties equivalent to des-pGlu1-brazzein (two times as sweet as the major form of brazzein isolated from fruit which contains pGlu1), and two were about twice as sweet as des-pGlu1-brazzein, Overall, the results suggest that two regions of the protein are critical for the sweetness of brazzein: a region that includes the N- and C-termini of the protein, which are located close to one another, and a region that includes the flexible loop around Arg43, (C) 2000 Academic Press.