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Accurate size-based protein localization from cryo-ET tomograms 从低温电子断层扫描图中准确定位蛋白质大小
IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-26 DOI: 10.1016/j.yjsbx.2024.100104
Weisheng Jin , Ye Zhou , Alberto Bartesaghi

Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within crowded cellular tomograms so that they can be aligned and averaged in 3D to improve resolution. While extensive work in 2D particle picking has been done in the context of single-particle cryo-EM, comparatively fewer strategies have been proposed to pick particles from 3D tomograms, in part due to the challenges associated with working with noisy 3D volumes affected by the missing wedge. While strategies based on 3D template-matching and deep learning are commonly used, these methods are computationally expensive and require either an external template or manual labelling which can bias the results and limit their applicability. Here, we propose a size-based method to pick particles from tomograms that is fast, accurate, and does not require external templates or user provided labels. We compare the performance of our approach against a commonly used algorithm based on deep learning, crYOLO, and show that our method: i) has higher detection accuracy, ii) does not require user input for labeling or time-consuming training, and iii) runs efficiently on non-specialized CPU hardware. We demonstrate the effectiveness of our approach by automatically detecting particles from tomograms representing different types of samples and using these particles to determine the high-resolution structures of ribosomes imaged in vitro and in situ.

低温电子断层扫描(cryo-ET)与子断层平均(STA)相结合,可以确定在细胞原生环境中以接近原子分辨率成像的蛋白质结构。粒子拾取是低温电子显微镜/STA 图像分析流水线中的一个重要步骤,它包括在拥挤的细胞断层图中定位蛋白质的位置,以便对它们进行三维对齐和平均,从而提高分辨率。虽然在单颗粒冷冻电子显微镜下进行了大量的二维颗粒拾取工作,但提出的从三维断层图中拾取颗粒的策略相对较少,部分原因是在处理受缺失楔形影响的噪声三维体积时面临挑战。虽然基于三维模板匹配和深度学习的策略很常用,但这些方法的计算成本很高,而且需要外部模板或人工标注,会使结果产生偏差,限制了其适用性。在这里,我们提出了一种基于尺寸的方法来从断层图中拾取粒子,这种方法快速、准确,而且不需要外部模板或用户提供的标签。我们将我们的方法与常用的基于深度学习的算法 crYOLO 进行了性能比较,结果表明我们的方法:i) 检测准确率更高;ii) 不需要用户输入标签或耗时的训练;iii) 可在非专用 CPU 硬件上高效运行。我们从代表不同类型样本的断层图中自动检测颗粒,并利用这些颗粒确定体外和原位成像核糖体的高分辨率结构,从而证明了我们方法的有效性。
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引用次数: 0
Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis 枯草芽孢杆菌中色氨酸生物合成的同源三叉戟形调控因子 Anti-TRAP 的溶液结构、动力学和四面体组装
IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-11 DOI: 10.1016/j.yjsbx.2024.100103
Craig A. McElroy , Elihu C. Ihms , Deepak Kumar Yadav , Melody L. Holmquist , Vibhuti Wadhwa , Vicki H. Wysocki , Paul Gollnick , Mark P. Foster

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

细胞生产色氨酸的代谢成本很高,而且受到严格调控。作为 yczA/rtpA 基因的产物,小型枯草芽孢杆菌锌结合抗 TRAP 蛋白(AT)通过 T-box 反凋亡机制上调无电荷 tRNATrp 的累积水平。AT 与非十聚体轴对称环形蛋白 TRAP(trp RNA 结合衰减蛋白)结合,从而阻止其与 trp 领导 RNA 结合。这就逆转了 TRAP 对 trp 操作子转录和翻译的抑制作用。AT 主要有两种对称的低聚物状态,一种是三聚体(AT3),具有三对折轴对称性;另一种是十二聚体(AT12),由四面体的三聚体组成,而只有三聚体形式才能结合并抑制 TRAP。我们应用本征质谱(nMS)和小角 X 射线散射(SAXS)以及分析超速离心(AUC)来监测 AT 的三聚体和十二聚体结构形式之间随 pH 值和浓度变化的平衡。此外,我们还利用溶液核磁共振(NMR)光谱确定了 AT3 的溶液结构,而对 AT 的两种低聚物形式进行的异核 15N 弛豫测量则让我们深入了解了具有结合活性的 AT3 和不具有结合活性的 AT12 的动态特性,这对 TRAP 的结合和抑制具有重要意义。
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引用次数: 0
Eliminating the missing cone challenge through innovative approaches 通过创新方法消除缺锥难题
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-06-01 DOI: 10.1016/j.yjsbx.2024.100102
Cody Gillman , Guanhong Bu , Emma Danelius , Johan Hattne , Brent L. Nannenga , Tamir Gonen

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.

微晶电子衍射(MicroED)已成为一种强大的技术,可用于揭示因晶体太小而无法进行 X 射线衍射的分子结构。然而,板状晶体在电子显微镜网格上始终保持平直方向时,会遇到一个重大障碍。如果平板的法线与晶格的轴线相关联,则可用于测量的晶体取向就会受到限制,因为晶体不能任意旋转。这就限制了可获取的信息,导致信息锥缺失。我们最近推出了一种名为悬滴结晶的新型结晶策略,并提出悬滴中的晶体可以有效解决首选晶体取向的难题。在这里,我们展示了悬滴法在两种结晶为薄板的样品(牛肝过氧化氢酶和 SARS-CoV-2 主要蛋白酶 (Mpro))中消除缺失锥的成功案例。事实证明,这种创新解决方案对于表现出系统性优选取向的晶体是不可或缺的,从而为通过 MicroED 进行结构测定开辟了新的可能性。
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引用次数: 0
Coordination of bilayer properties by an inward-rectifier K+ channel is a cooperative process driven by protein-lipid interaction 内向整流 K+ 通道对双分子层特性的协调是一个由蛋白质-脂质相互作用驱动的合作过程
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-06-01 DOI: 10.1016/j.yjsbx.2024.100101
Evan J. van Aalst , Maryam Yekefallah , Roy A. M. van Beekveld , Eefjan Breukink , Markus Weingarth , Benjamin J. Wylie

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.

生物膜的物理特性直接或间接地影响着生物过程。然而,由于膜蛋白、单个脂质和膜结构之间存在相互影响,因此很难评估膜蛋白和完整膜蛋白之间的相互作用。我们之前利用固态核磁共振(SSNMR)研究发现,细菌内向整流 K+ 通道 KirBac1.1 通过拴系阴离子脂质而核化双分子层有序化和微域的形成。相反,这些脂质与阳离子残基合作结合,激活通道并启动 K+ 通量。然而,控制脂质合作负载与双分子层有序化之间关系的机制细节尚不清楚。为了进行研究,我们生成了含有不同浓度 13C 锂化磷脂酰甘油 (PG) 脂质的 KirBac1.1 样品,并使用有序的全反式 (AT) 和无序的反式-高切 (TG)酰构象作为双分子层动态的标记,获得了一整套 SSNMR 1D 温度序列实验。我们观察到随着 PG 浓度的增加,AT 有序信号增加,TG 无序信号减少,双分子层熔融温度升高。此外,我们还发现了有序化与 PG 脂类直接结合之间的合作关系,这表明 KirBac1.1 驱动的双分子层有序化和微域形成是一个经典的合作希尔型过程,由 PG 脂类直接结合驱动并以其为前提。我们的研究结果为蛋白质和脂质如何共同促成脂膜超分子双分子层异质性提供了独特的机理见解。
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引用次数: 0
Optimizing NMR fragment-based drug screening for membrane protein targets 优化基于核磁共振片段的膜蛋白靶点药物筛选
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-06-01 DOI: 10.1016/j.yjsbx.2024.100100
Geoffrey C. Li , Manuel A. Castro , Thilini Ukwaththage, Charles R. Sanders

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR—especially human membrane proteins—and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

核磁共振光谱法将配体与靶标的弱结合检测与结合位点的结构图绘制结合起来,在基于片段的药物发现中发挥了关键作用。基于核磁共振的片段筛选已成功应用于许多可溶性蛋白质靶点,但只应用于数量有限的膜蛋白,尽管事实上许多药物靶点都是膜蛋白。部分原因是难以制备 NMR 所需的膜蛋白--尤其是人类膜蛋白--以及膜蛋白样品溶液 NMR 光谱固有的复杂性,这需要加入膜模拟剂,如胶束、纳米盘或双胞。在此,我们开发了一种可通用的方案,利用 NMR 对膜蛋白进行基于片段的筛选。我们采用了两个人类膜蛋白靶标,它们都在完全质子化的洗涤剂胶束中:淀粉样前体蛋白 C99 的单通道 C 端结构域和外周髓鞘蛋白 22 (PMP22) 的四跨结构域。我们确定了这两种药物的最佳 NMR 采集参数、蛋白质浓度、蛋白质与胶束的比率以及筛选样品中 D6-DMSO 的浓度上限。此外,我们还利用优化条件对板式分子片段混合物库进行了初步筛选,并确定了可选择性结合到相应靶蛋白的命中化合物。希望本文介绍的方法能对发现靶向膜蛋白的先导化合物的现有方法起到补充作用。
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引用次数: 0
The parabasal filaments of Trichomonas vaginalis: A new filament and observations using 0.8 nm-resolution scanning electron microscopy 阴道毛滴虫的副基质丝:一种新的丝状物和使用 0.8 纳米分辨率扫描电子显微镜的观察结果
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-03-06 DOI: 10.1016/j.yjsbx.2024.100099
Sharmila Fiama das Neves Ortiz , Raphael Verdan , Gustavo Miranda Rocha , Kildare Miranda , Marlene Benchimol

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common nonviral sexually transmitted infection worldwide, with an estimated 260 million new cases annually. T. vaginalis contains organelles common to all eukaryotic cells, uncommon cell structures such as hydrogenosomes, and a complex and elaborate cytoskeleton constituting the mastigont system. The mastigont system is mainly formed by several proteinaceous structures associated with basal bodies, the pelta-axostylar complex made of microtubules, and striated filaments named the costa and the parabasal filaments (PFs). Although the structural organization of trichomonad cytoskeletons has been analyzed using several techniques, observation using a new generation of scanning electron microscopes with a resolution exceeding 1 nm has allowed more detailed visualization of the three-dimensional organization of the mastigont system. In this study, we have investigated the cytoskeleton of T. vaginalis using a diverse range of scanning probe microscopy techniques, which were complemented by electron tomography and Fast-Fourier methods. This multi-modal approach has allowed us to characterize an unknown parabasal filament and reveal the ultrastructure of other striated fibers that have not been published before. Here, we show the differences in origin, striation pattern, size, localization, and additional details of the PFs, thus improving the knowledge of the cell biology of this parasite.

阴道毛滴虫是滴虫病的病原体,是全球最常见的非病毒性传播感染,估计每年新增病例 2.6 亿例。阴道毛滴虫含有所有真核细胞共有的细胞器、不常见的细胞结构(如氢小体)以及复杂而精细的细胞骨架,这些构成了阴道毛滴虫系统(mastigont system)。mastigont系统主要由与基底体、由微管组成的骨盆-轴柱复合体以及名为costa和parabasal丝(PFs)的横纹丝相关的几种蛋白质结构组成。虽然已经使用多种技术分析了毛滴虫细胞骨架的结构组织,但使用分辨率超过 1 nm 的新一代扫描电子显微镜进行观察,可以更详细地观察到丝束系统的三维组织。在这项研究中,我们使用了多种扫描探针显微镜技术,并辅以电子断层扫描和快速傅立叶方法,对阴道蓟马的细胞骨架进行了研究。这种多模式方法使我们得以描述一种未知副基丝的特征,并揭示了以前未发表过的其他横纹纤维的超微结构。在这里,我们展示了副寄生虫丝在起源、纹路模式、大小、定位和其他细节方面的差异,从而增进了对这种寄生虫细胞生物学的了解。
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引用次数: 0
Magic-angle spinning NMR structure of Opa60 in lipid bilayers Opa60 在脂质双分子层中的魔角旋转 NMR 结构
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-02-21 DOI: 10.1016/j.yjsbx.2024.100098
Marcel C. Forster, Kumar Tekwani Movellan, Eszter E. Najbauer, Stefan Becker, Loren B. Andreas

Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.

在此,我们利用质子检测的魔角旋转核磁共振(MAS NMR)技术报告了 Opa60 在脂质双层中的结构。与之前报道的去垢剂结构相比,包括近原生寡糖脂质在内的制备方法揭示了稳定的跨膜β桶的一致图像,结构区域略有增加。无法检测到已知的与宿主蛋白相互作用的大型可变环,这证实了它们即使在脂质双分子层环境中也是动态的。该结构为研究 Opa60 在淋球菌感染中的功能作用提供了一个起点,据了解,这种作用涉及与宿主蛋白的相互作用。同时,它还展示了质子检测方法在通过 MAS NMR 以原子分辨率确定膜蛋白结构方面的最新进展。
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引用次数: 0
Grafting the ALFA tag for structural studies of aquaporin Z 接枝 ALFA 标签用于水蒸发蛋白 Z 的结构研究
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-02-02 DOI: 10.1016/j.yjsbx.2024.100097
Lauren Stover , Hanieh Bahramimoghaddam , Lie Wang , Samantha Schrecke , Gaya P. Yadav , Ming Zhou , Arthur Laganowsky

Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate structure determination of membrane proteins, such as the use of antibodies. Here, we graft onto AqpZ the ALFA tag (AqpZ-ALFA), an alpha helical epitope, to make use of the high-affinity anti-ALFA nanobody (nB). Native mass spectrometry reveals the AqpZ-ALFA fusion forms a stable, 1:1 complex with nB. Single-particle cryogenic electron microscopy studies reveal the octameric (AqpZ-ALFA)4(nB)4 complex forms a dimeric assembly and the structure was determined to 1.9 Å resolution. Dimerization of the octamer is mediated through stacking of the symmetrically bound nBs. Tube-like density is also observed, revealing a potential cardiolipin binding site. Grafting of the ALFA tag, or other epitope, along with binding and association of nBs to promote larger complexes will have applications in structural studies and protein engineering.

水蒸发蛋白 Z(AqpZ)是一种细菌水通道,形成四聚体复合物,与许多其他膜蛋白一样,其活性受脂类调节。为了便于确定膜蛋白的结构,人们开发了多种方法,如使用抗体。在这里,我们在 AqpZ 上嫁接了 ALFA 标签(AqpZ-ALFA),这是一个阿尔法螺旋表位,从而利用了高亲和力的抗 ALFA 纳米抗体(nB)。本征质谱显示,AqpZ-ALFA 融合体与 nB 形成了稳定的 1:1 复合物。单颗粒低温电子显微镜研究显示,八聚体(AqpZ-ALFA)4(nB)4 复合物形成二聚体组装,其结构的分辨率达到 1.9 Å。八聚体的二聚化是通过对称结合的 nBs 的堆积来介导的。还观察到管状密度,揭示了一个潜在的心磷脂结合位点。嫁接 ALFA 标记或其他表位,以及 nBs 的结合和关联以促进更大的复合物,将在结构研究和蛋白质工程中得到应用。
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引用次数: 0
Structure of biomimetic casein micelles: Critical tests of the hydrophobic colloid and multivalent-binding models using recombinant deuterated and phosphorylated β-casein 仿生酪蛋白胶束的结构:使用重组氘化和磷酸化 β -酪蛋白对疏水胶体和多价结合模型进行关键测试
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-22 DOI: 10.1016/j.yjsbx.2024.100096
Jared K. Raynes , Jitendra Mata , Karyn L. Wilde , John A. Carver , Sharon M. Kelly , Carl Holt

Milk contains high concentrations of amyloidogenic casein proteins and is supersaturated with respect to crystalline calcium phosphates such as apatite. Nevertheless, the mammary gland normally remains unmineralized and free of amyloid. Unlike κ-casein, β- and αS-caseins are highly effective mineral chaperones that prevent ectopic and pathological calcification of the mammary gland. Milk invariably contains a mixture of two to five different caseins that act on each other as molecular chaperones. Instead of forming amyloid fibrils, several thousand caseins and hundreds of nanoclusters of amorphous calcium phosphate combine to form fuzzy complexes called casein micelles. To understand the biological functions of the casein micelle its structure needs to be understood better than at present. The location in micelles of the highly amyloidogenic κ-casein is disputed. In traditional hydrophobic colloid models, it, alone, forms a stabilizing surface coat that also determines the average size of the micelles. In the recent multivalent-binding model, κ-casein is present throughout the micelle, in intimate contact with the other caseins. To discriminate between these models, a range of biomimetic micelles was prepared using a fixed concentration of the mineral chaperone β-casein and nanoclusters of calcium phosphate, with variable concentrations of κ-casein. A biomimetic micelle was also prepared using a highly deuterated and in vivo phosphorylated recombinant β-casein with calcium phosphate and unlabelled κ-casein. Neutron and X-ray scattering experiments revealed that κ-casein is distributed throughout the micelle, in quantitative agreement with the multivalent-binding model but contrary to the hydrophobic colloid models.

牛奶中含有高浓度的致淀粉样蛋白的酪蛋白,并含有过饱和的结晶钙磷酸盐,如磷灰石。不过,乳腺通常不会矿化,也不含淀粉样蛋白。与κ-酪蛋白不同,β-和α-S-酪蛋白是高效的矿物质伴侣,可防止乳腺发生异位和病理性钙化。牛奶中无一例外地含有两到五种不同酪蛋白的混合物,这些酪蛋白相互之间起着分子伴侣的作用。几千个酪蛋白和数百个无定形磷酸钙纳米簇结合成模糊的复合物,称为酪蛋白胶束,而不是形成淀粉样纤维。要了解酪蛋白胶束的生物功能,就必须对其结构有更深入的了解。高度淀粉样化的κ-酪蛋白在胶束中的位置存在争议。在传统的疏水胶体模型中,κ-酪蛋白单独形成一层稳定的表面包膜,这也决定了胶束的平均大小。在最新的多价结合模型中,κ-酪蛋白存在于整个胶束中,与其他酪蛋白亲密接触。为了区分这些模型,我们使用固定浓度的矿物伴侣β-酪蛋白和纳米磷酸钙簇以及不同浓度的κ-酪蛋白制备了一系列仿生胶束。此外,还利用高度氚化和体内磷酸化的重组β-酪蛋白与磷酸钙和未标记的κ-酪蛋白制备了一种仿生物胶束。中子和 X 射线散射实验显示,κ-酪蛋白分布在整个胶束中,这与多价结合模型在数量上一致,但与疏水胶体模型相反。
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引用次数: 0
Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR 基于蛋白胨的可变脂质成分大圆盘,用于通过定向样品固态核磁共振进行膜蛋白结构研究
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-04 DOI: 10.1016/j.yjsbx.2023.100095
Azamat R. Galiakhmetov, Adit A. Shah, Addison Lane, Carolynn M. Davern, Caroline Proulx, Alexander A. Nevzorov

Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface.

将固态核磁共振(NMR)与磁性排列的盘状脂质模拟物相结合,可以在生理温度下研究膜蛋白在平面、富脂双层环境中的构象。我们最近证明了由 DMPC 脂质和蛋白胨带组成的大圆盘的普遍适用性,其磁性排列和 NMR 光谱分辨率可与含洗涤剂的双胞相媲美或更胜一筹。在此,我们报告了由齐聚物和带负电荷的脂质混合物(DMPC/DMPG,摩尔比为 85%-15%)组成的蛋白胨基大圆盘在磁排列和 NMR 分辨率方面的显著改进。由于圆盘之间的稳定静电斥力可能会产生较高的定向有序参数,因此产生的线宽锐化了约 30%。此外,长链脂质 DPPC 也能形成高度排列整齐、不含洗涤剂的大圆盘。有趣的是,Pf1 在两种脂质模拟物中的光谱几乎没有区别,这意味着整体跨膜螺旋倾斜可能不仅受疏水匹配的影响,还可能受膜界面侧翼赖氨酸和精氨酸残基的相互作用的影响。
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Journal of Structural Biology: X
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