Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
|
Author:
Date:
2017-09-05
[Abstract] Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).
[摘要] 研究蛋白质的液 - 液相分离(LLPS)提供了无膜细胞器的生物发生和疾病中病理蛋白聚集的关键见解。 我们已经建立了诱导富含精氨酸的肽的相分离的方案,这允许研究其分子决定因素和动力学(Boeynaems等,2017)。 【背景】富含精氨酸的无序结构域通常存在于RNA结合蛋白中,包括与神经变性疾病相关的蛋白(例如FUS,FMRP,hnRNPA1)(Varadi等人,2015; Boeynaems等,2017)。 在携带C9orf72重复扩增的肌萎缩性侧索硬化患者中也产生有毒的富含精氨酸的重复肽(即polyGR和polyPR)(Kwon等,2014; Mizielinska等,2014; Varadi等,2015; Boeynaems et 等等,2016)。 (Kato et al。,2012; Burke et al。,2015; Lin et al。,2015; Molliex et al。,2015; Patel et al。,2015),虽然已经研究了不带电的低复杂度域的相分离, 开发了该方案来测试富含精氨酸的结构域是否也有助于相分离。 协议的示意图可以在图1中看到。
|
|
Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
|
Author:
Date:
2015-10-20
[Abstract] Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods ...
[摘要] 在异质发酵细菌物种,例如otakiensis的乳酸杆菌(Lactobacillus otakiensis)的培养基中观察到D-亮氨酸,D-异亮氨酸 - 异亮氨酸和D-缬氨酸的显着积累。 otakiensis)JCM 15040.从细菌细胞中纯化导致这种积累的消旋酶,异亮氨酸2-差向异构酶。这是首次报道的在乳酸菌中这种D-支链氨基酸的产生的观察结果,以及在任何生物体中具有异亮氨酸2-差向异构酶活性的消旋酶的第一个实例。在所述的方案中,我们介绍从L中纯化该蛋白质的方法。因为没有鉴定到对该酶具有高亲和力的特异性配体,所以使用硫酸铵级分,四种类型的柱层析和制备型Native-PAGE,不使用亲和柱层析进行纯化。我们希望协议将提供有用的信息用于纯化不能容易地使用亲和柱层析纯化的酶。
|
|
Analysis of L- and D-Amino Acids Using UPLC
|
Author:
Date:
2014-09-05
[Abstract] With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in ...
[摘要] 除了甘氨酸,α-氨基酸具有光学活性,并且可以形成每个氨基酸的两种光学异构体(L-和D-)。分析技术的最新发展表明,几种游离D-氨基酸如D-天冬氨酸,D-丝氨酸和D-丙氨酸存在于包括人在内的许多种生物中,并且具有生物学上的重要作用。 D-天冬氨酸调节动物和人的生殖活性。 D-丝氨酸作为Nε-甲基-D-天冬氨酸受体的共激动剂,其介导谷氨酸能神经传递。 D-丙氨酸在甲壳类动物和软体动物中起到类似渗透物的作用。在该协议中,我们描述了使用超高效液相色谱(UPLC)分析L-和D-氨基酸的方法。为了分析D-和L-氨基酸,首先将对映异构体转化为非对映异构体(非对映异构体是与物镜和镜像无关的立体异构体,并且不是对映异构体),使用前柱衍生化,邻苯二甲醛加上Nε - 酰化的半胱氨酸(Nε - 乙酰基-L-半胱氨酸或Nε - 叔丁基氧羰基-L 。所得衍生物是荧光非对映异构体。然后使用UPLC在十八烷基硅烷基固定相上分离所得荧光异吲哚衍生物,并通过包括在UPLC系统中的荧光检测器检测荧光。使用该方法,可以分析16种D-氨基酸。
|
|