Zwick M B, Shen J, Scott J K
Department of Molecular Biology and Biochemistry, Simon Fraser University Burnaby, BC, Canada.
J Mol Biol. 2000 Jul 7;300(2):307-20. doi: 10.1006/jmbi.2000.3850.
Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.
丝状噬菌体展示的肽库已被证明是发现新型肽激动剂、拮抗剂和表位模拟物的有力工具。大多数噬菌体展示的肽与次要外壳蛋白pIII或主要外壳蛋白pVIII的N端融合。我们在此报告，作为pVIII的N端融合物展示的含半胱氨酸残基的肽可在噬菌体外壳上形成二硫键连接的同二聚体。从含有一个或两个固定半胱氨酸残基的文库中随机选择噬菌体克隆，并通过SDS-PAGE分析检测肽-pVIII同二聚体的存在，该分析涉及用还原剂或硫醇修饰剂预处理噬菌体。对于所有重组肽含有单个半胱氨酸残基的噬菌体，相当一部分肽-pVIII分子以二聚体形式展示在噬菌体外壳上。在几乎所有能可靠观察到两种形式的情况下，二聚体形式都比单体形式更丰富。偶尔，含有两个半胱氨酸残基的肽也会形成二聚体。这些结果表明，对于给定的带有单个半胱氨酸残基的pVIII展示肽，无论其序列如何，相当一部分肽（>40%）都会二聚化；然而，序列限制可能决定所有肽是否都会二聚化。同样，含有两个半胱氨酸残基的肽只是偶尔形成分子间二硫键而不是分子内二硫键；这表明序列限制也可能决定二聚化与环化。对表达与含有单个半胱氨酸残基的肽融合的pVIII的细胞的膜进行蔗糖梯度分析表明，二聚体pVIII在组装到噬菌体上之前就存在于细胞中。根据噬菌体外壳的结构和组装的现有模型讨论了肽-pVIII同二聚体的模型。噬菌体表面肽的共价结合产生的独特二级结构表明同二聚体和异二聚体肽库作为生物活性肽新来源的作用。
