Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, United States.
Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, United States.
Protein Eng Des Sel. 2024 Jan 29;37. doi: 10.1093/protein/gzae006.
Proline-rich antimicrobial peptides (PrAMPs) are attractive antibiotic candidates that target gram-negative bacteria ribosomes. We elucidated the sequence-function landscape of 43 000 variants of a recently discovered family member, Tur1a, using the validated SAMP-Dep platform that measures intracellular AMP potency in a high-throughput manner via self-depletion of the cellular host. The platform exhibited high replicate reproducibility (ρ = 0.81) and correlation between synonymous genetic variants (R2 = 0.93). Only two segments within Tur1a exhibited stringent mutational requirements to sustain potency: residues 9YLP11 and 19FP20. This includes the aromatic residue in the hypothesized binding domain but not the PRP domain. Along with unexpected mutational tolerance of PRP, the data contrast hypothesized importance of the 1RRIR4 motif and arginines in general. In addition to mutational tolerance of residue segments with presumed significance, 77% of mutations are functionally neutral. Multimutant performance mainly shows compounding effects from removed combinations of prolines and arginines in addition to the two segments of residues showing individual importance. Several variants identified as active from SAMP-Dep were externally produced and maintained activity when applied to susceptible species exogenously.
富含脯氨酸的抗菌肽（PrAMPs）是一种有吸引力的抗生素候选物，其作用靶标是革兰氏阴性菌核糖体。我们使用经过验证的 SAMP-Dep 平台，阐明了最近发现的一种成员 Tur1a 的 43000 种变体的序列-功能图谱，该平台通过细胞宿主的自我耗竭，以高通量的方式测量细胞内 AMP 效力。该平台表现出高复制重现性（ρ=0.81）和同义遗传变异体之间的相关性（R2=0.93）。只有 Tur1a 内的两个片段表现出严格的突变要求以维持效力：残基 9YLP11 和 19FP20。这包括假设的结合域中的芳香残基，但不包括 PRP 结构域。与假设的结合域的重要性以及精氨酸的重要性相反，PRP 具有出人意料的突变容忍性。除了假定具有重要意义的残基片段的突变容忍性之外，77%的突变是功能中性的。多突变体的性能主要表现为除了两个表现出个体重要性的残基片段之外，去除脯氨酸和精氨酸的组合所产生的复合效应。从 SAMP-Dep 中鉴定出的几种具有活性的变体被外部产生，并在对易感物种进行外源应用时保持活性。
