Jing Weiguo, Demcoe Alistair R, Vogel Hans J
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Bacteriol. 2003 Aug;185(16):4938-47. doi: 10.1128/JB.185.16.4938-4947.2003.
Puroindoline a, a wheat endosperm-specific protein containing a tryptophan-rich domain, was reported to have antimicrobial activities. We found that a 13-residue fragment of puroindoline a (FPVTWRWWKWWKG-NH(2)) (puroA) exhibits activity against both gram-positive and gram-negative bacteria. This suggests that puroA may be a bactericidal domain of puroindoline a. PuroA interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the microbicidal effect of puroA may be due to interactions with bacterial membranes. A variety of biophysical and biochemical methods, including fluorescence spectroscopy and microcalorimetry, were used to examine the mode of action of puroA. These studies showed that puroA is located at the membrane interface, probably due to its high content of Trp residues that have a high propensity to partition into the membrane interface. The penetration of these Trp residues in negatively charged phospholipid vesicles resembling bacterial membranes was more extensive than the penetration in neutral vesicles mimicking eukaryotic membranes. Peptide binding had a significant influence on the phase behavior of the former vesicles. The three-dimensional structure of micelle-bound puroA determined by two-dimensional nuclear magnetic resonance spectroscopy indicated that all the positively charged residues are oriented close to the face of Trp indole rings, forming energetically favorable cation-pi interactions. This characteristic, along with its well-defined amphipathic structure upon binding to membrane mimetic systems, allows puroA to insert more deeply into bacterial membranes and disrupt the regular membrane bilayer structure.
麦醇溶蛋白a是一种小麦胚乳特异性蛋白，含有一个富含色氨酸的结构域，据报道具有抗菌活性。我们发现麦醇溶蛋白a的一个13个残基的片段（FPVTWRWWKWWKG-NH₂）（puroA）对革兰氏阳性菌和革兰氏阴性菌均有活性。这表明puroA可能是麦醇溶蛋白a的杀菌结构域。puroA与带负电荷的磷脂囊泡强烈相互作用，并诱导这些囊泡有效释放染料，这表明puroA的杀菌作用可能是由于与细菌膜的相互作用。我们使用了多种生物物理和生化方法，包括荧光光谱法和微量量热法，来研究puroA的作用模式。这些研究表明，puroA位于膜界面，这可能是由于其高含量的色氨酸残基，这些残基很容易分配到膜界面。这些色氨酸残基在类似于细菌膜的带负电荷的磷脂囊泡中的渗透比在模拟真核细胞膜的中性囊泡中的渗透更广泛。肽结合对前者囊泡的相行为有显著影响。通过二维核磁共振光谱法测定的与胶束结合的puroA的三维结构表明，所有带正电荷的残基都靠近色氨酸吲哚环的表面排列，形成能量上有利的阳离子-π相互作用。这一特性，连同其在与膜模拟系统结合时明确的两亲结构，使puroA能够更深入地插入细菌膜并破坏规则的膜双层结构。
