Department of Biological Sciences, Towson University, Towson, MD, United States of America.
PLoS One. 2018 Jun 18;13(6):e0198699. doi: 10.1371/journal.pone.0198699. eCollection 2018.
Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.
小分子蛋白质是一个新的且不断发展的研究领域。许多已鉴定的小分子蛋白质由单个疏水性α-螺旋组成，其有限的氨基酸序列的功能要求还不是很清楚。一种疏水性小分子蛋白质 CydX 已被证明是大肠杆菌细胞色素 bd 氧化酶复合物的一个组成部分，并且是酶功能所必需的。为了研究小分子蛋白质的序列特异性，使用在野生型基因座上从大肠杆菌染色体表达的突变等位基因对小分子蛋白质 CydX 进行了丙氨酸扫描诱变。对产生的突变株进行了 CydX 功能测定。没有一个单一的氨基酸需要维持对β-巯基乙醇的野生型抗性。然而，10 个氨基酸块的取代表明，蛋白质的 N 末端对于野生型 CydX 活性是必需的。一系列的双突变体表明，N 末端的多个突变导致体内β-巯基乙醇敏感性。三突变体显示出体内和体外表型。这些数据共同提供了证据，表明 CydX 具有高度的功能可塑性，其中多个氨基酸可能协同作用以促进 CydX 功能。
