嗅觉系统能够检测和区分数千种在繁殖中起关键作用的环境气味,包括信息素在内的社会行为影响了经典事件。膜秘书气味结合蛋白(OBP)是可溶性脂质运载蛋白,位于哺乳动物的鼻膜中。它们在鼻上皮内结合并携带气味物质至推定的嗅觉跨膜受体(ORs)。尚未利用OBP来开发合适的检测发情的技术,据报道这是水牛的一项艰巨任务。在本研究中,使用分子生物学和蛋白质工程方法,我们从水牛鼻上皮气味结合蛋白(bnOBP)中克隆了六种新型OBP亚型。此外,开发了三维模型和分子对接,动力学实验是通过计算机模拟方法进行的。特别是,我们在OBP1a中发现了四个残基(Phe104,Phe134,Phe69和Asn118),这有助于对两种性信息素的有利相互作用,特别是油酸和对甲酚。我们在雌性水牛尿液中的大肠杆菌中表达了该蛋白,并通过荧光猝灭研究进行了验证,以显示OBP1a与油酸和对甲酚的结合亲和力相似。通过使用结构数据,结合特异性还通过4个残基的定点诱变,然后进行体外结合测定来验证.我们的结果使我们能够更好地了解水牛不同鼻上皮OBP同工型的功能。他们还提高了对嗅觉蛋白和气味剂之间相互作用的理解,以开发高度选择性的生物传感设备,用于水牛发情的非侵入性检测。由RamaswamyH.Sarma沟通。
The olfactory system is capable of detecting and distinguishing thousands of environmental odorants that play a key role in reproduction, social behaviours including pheromones influenced classical events. Membrane secretary odorant binding proteins (OBPs) are soluble lipocalins, localized in the nasal membrane of mammals. They bind and carry odorants within the nasal epithelium to putative olfactory transmembrane receptors (ORs). OBP has not yet been exploited to develop a suitable technique to detect oestrus which is being reported as a difficult task in buffalo. In the present study, using molecular biology and protein engineering approaches, we have cloned six novel OBP isoforms from buffalo nasal epithelium odorant-binding proteins (bnOBPs). Furthermore, 3 D models were developed and molecular-docking, dynamics experiments were performed by in silico approaches. In particular, we found four residues (Phe104, Phe134, Phe69 and Asn118) in OBP1a, which contributed to favourable interactions towards two sex pheromones, specifically oleic acid and p-cresol. We expressed this protein in Escherichia coli from female buffalo urine and validated through fluorescence quenching studies to show similar strong binding affinities of OBP1a to oleic acid and p-cresol. By using structural data, the binding specificity was also verified by site-directed mutagenesis of the four residues followed by in vitro binding assays. Our results enable us to better understand the functions of different nasal epithelium OBP isoforms in buffaloes. They also lead to improved understanding of the interaction between olfactory proteins and odorants to develop highly selective biosensing devices for non-invasive detection of oestrus in buffaloes. Communicated by Ramaswamy H. Sarma.