Published as part of Chemical Reviews virtual special issue “Molecular Crowding”. The term “macromolecular crowding” was originally introduced to take into account the impact of the macromolecular environment surrounding proteins on their stability and reaction rates when in the highly volume-occupied or “crowded” solutions, as they experience in the cell or biological fluids. This concept promoted studies aiming at reproducing the intracellular environment in which biochemical reactions occur, more faithfully than what is usually considered in the typical test tube. Crowding nonspecifically enhances reactions, leading to the reduction of total excluded volume, such as the formation of macromolecular complexes in solution and compaction or folding of proteins. As the cell interior, even of the simplest prokaryotic systems, contains a variety of microenvironments, with spatial and compositional heterogeneity, the potential implications of additional specific and nonspecific interactions, in addition to volume exclusion, including surface adsorption and macromolecular partitioning, have to be considered to understand macromolecular reactivity in vivo. These challenges have expanded the field, and a substantial number of experimental and simulation studies have been conducted with the aim of answering, at least in part, the biologically relevant questions on how much and why macromolecular reactions in cells differ from those measured in test tubes. This virtual thematic issue of Chemical Reviews aims to discuss how the concept of molecular crowding has evolved within the last two decades, touching new complementary territories, and also as a result of the introduction of new approaches employed in crowding studies which have offered further potentialities to study crowding. After a historical introduction to the subject and an analysis of the evolution of the crowding concept over time, Alfano et al. (1) introduced various aspects covered in depth in other reviews of this issue. In addition to the well-known biological themes of protein stability and protein activity, the article deals with the origin of crowding from the perspective of colloidal and polymer physics. They also considered crowding effects on phase transitions, including those related to protein aggregation, amyloid formation, and phase-separated biomolecular condensation. They also discussed in detail the value of various environmental conditions and using different crowders. Careful selection of appropriate crowding agents and their concentrations is crucial for mimicking the natural crowded environment in experimental setups. The ultimate goal of crowding studies remains to elucidate its impact on biological function. To this end, Monterroso et al. (2) analyzed how macromolecular crowding, phase separation, and physicochemical homeostasis orchestrate bacterial intracellular organization and essential cell-cycle processes. They first discussed the various factors influencing the structure of the cytoplasm─including crowding and physicochemical parameters such as pH, ionic strength, and energy status─and compared the supramolecular structures found in bacteria with those of eukaryotic cells. Then, they analyzed the impact of crowding and phase separation on the bacterial nucleoid organization, and the regulation of chromosome replication, segregation, and bacterial cell division processes, and the implications of the membrane of these processes. Finally, they discussed how biomolecular condensates driven by phase separation can relate to the modulation of bacterial fitness. Traditionally, the spotlight on the effect of crowding on biomacromolecules has been almost exclusively pointed on proteins. However, the role played by nucleic acids is also crucial, as argued by Zacco et al., (3) who discussed the impact of crowding on RNA. The authors explored the pivotal role played by RNA sequence, structure, and chemical modifications in granules or biological condensates caused by crowding events. In addition to exploring the role played by RNA under physiological conditions that are then functionally important, the authors investigate instances in which crowding deviates from its intended function, leading to pathological consequences. They also evaluated the methodologies employed to decipher the composition of RNA granules, offering a comprehensive overview of the techniques used to characterize them. Altogether, the authors offered a multifaceted understanding of the world of RNA-mediated condensates. In the current thematic issue dedicated to molecular crowding, the contributions based on experimental approaches are flanked by the impactful paper by Grassmann et al., (4) who discussed theoretical and computational approaches that allow the modeling of biological systems to guide and complement experiments and can thus significantly advance the investigation, and possibly the predictions, of protein–protein interactions in the crowded environment of the cell cytoplasm. The authors explored topics such as statistical mechanics for lattice simulations, hydrodynamic interactions, diffusion processes in high-viscosity environments, and several methods based on molecular dynamics simulations. Always taking the opportunity of exploring the wide spectrum of topics covered at present by the concept of crowding, Olgenblum et al. (5) focused on a particular type of stress that can be faced by cells: the desiccation stress. Faced with this stress, many organisms deploy strategies to maintain the integrity of their cellular components. Amorphous glassy media present general strategies for protecting against drying. They reviewed these strategies and the proposed molecular mechanisms to explain protein protection in a vitreous matrix under conditions of low hydration. In the exploration of the diversity of topics covered by crowding, Peters et al. (6) tackled crowding under extreme conditions. According to the authors, it is becoming increasingly clear how important it is to take the environment into account if we are to shed light on biological function under various external conditions. Many biosystems thrive under extreme conditions, including the deep sea and subseafloor crust, and can take advantage of some of the effects of crowding. These relationships were studied in recent years using various biophysical techniques, including neutron and X-ray scattering, calorimetry, and FTIR, UV–vis, and fluorescence spectroscopies. Combining knowledge of the structure and conformational dynamics of biomolecules under extreme conditions, such as temperature, high hydrostatic pressure, and high salinity, Peters et al. highlighted the importance of considering the results in the context of the environment. Overall, the new technical and theoretical approaches allowed, among others, by the new generation of synchrotron radiation and by more powerful and better focused computational methods, are promoting new and compelling science, hopefully informing the next generation of researchers and encouraging them to explore further avenues that could conceive novel ways to design experiments that more faithfully model the intracellular environment. Annalisa Pastore is a structural biologist with more than 35 years’ experience in protein structure determination and a strong interest in protein stability, folding, and misfolding. After her masters and Ph.D. in Chemistry at the University Federico II of Naples, she spent a postdoc at Oxford University and ETH, Zurich. In 1988, she moved to the European Molecular Biology Laboratory (EMBL) in Heidelberg, where she started the first laboratory of Bio Nuclear Magnetic Resonance at the EMBL. In 1997–2013, she worked at the Medical Research Council London, focusing on both muscle proteins and proteins related to neurodegeneration. From 2013 to 2021, she worked at the Wohl Institute for Neuroscience of King’s College London. She is currently affiliated with King’s College London, Imperial College London, and Elettra Sincrotrone Trieste. Germán Rivas Caballero undertook doctoral training in the lab of José Gonzalez-Rodríguez (Physical Chemistry Institute, CSIC, Madrid, Spain). He received his Ph.D. in Chemistry in 1989 from Autonomous University of Madrid, Spain and then undertook postdoctoral training in the laboratories of Allen Minton (NIH, 1990–1992) and Jurgen Engel (1993, Biozentrum, University of Basel, Switzerland). He has worked since 1994 at the CIB Margarita Salas, CSIC, Madrid, and has been a group leader since 1996 and then a CSIC Research Professor since 2015. He has devoted his scientific career to studying multiprotein systems whose elements dynamically interact to organize functional cellular machines involved in essential processes. During his postdoctoral time in Minton’s lab, he realized the impact of the local microenvironment on the functional energetics of macromolecular associations in physiological (crowded) environments. He and his co-workers developed unique biophysical methods to study protein associations under crowding conditions mimicking the cell interior, allowing them to experimentally demonstrate that excluded volume effects due to crowding can significantly affect the mode and extent of protein association. For the last 25 years, the Rivas laboratory has explored the biochemical mechanisms governing the functional interactions of the bacterial division machinery (the divisome) to reconstruct with a bottom up approach simplified versions of the divisome in controlled cell-like environments. Piero Andrea Temussi (PAT) is a structural biologist who has been Professor of Chemistry at the Federico II University of Naples from 1963 to 2010. In his early career, he was a collaborator of Paolo Corradini and the Nobel Laureate Giulio Natta, who introduced stereotactic polymerization. PAT is one of the first two scientists, together with Anna Laura Segre, who introduced in the 70s biological nuclear magnetic resonance (NMR) spectroscopy in Italy in the early days of this technique, after a postdoctoral experience in the group of Prof. Herbert S. Gutowski in the University of Illinois Urbana–Champaign. Gutowsky was the first scientist to apply NMR methods to the field of chemistry. PAT was also a visiting scientist in the group of Shneinor Lifson, a highly reputed Israeli chemical physicist, Scientific Director of the Weizmann Institute of Science, and a founder of the Open University of Israel. Lifson is best known for his consistent force field method, one of the major theories behind 3D computer modeling of large molecules. Through his work in Italy, PAT is the promoter and author of compelling research on bioactively relevant peptides such as aspartame and several types of opioids. He is the author of more than 200 peer reviewed articles, also in high visibility journals. He was visiting professor at King’s College London (KCL) from 2015 to 2021. In 2019 he became a member of the Academia Europaea. His current scientific interests range from protein folding and stability to cold denaturation, crowding, and the molecular understanding of taste. This article references 6 other publications. This article has not yet been cited by other publications. This article references 6 other publications.

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简介：分子拥挤


作为《化学评论》虚拟特刊“分子拥挤”的一部分发表。 “大分子拥挤”一词最初是为了考虑蛋白质周围的大分子环境对其稳定性和反应速率的影响而引入的，当它们处于高体积占据或“拥挤”的溶液中时，就像它们在细胞或生物液体中所经历的那样。这一概念促进了旨在再现发生生化反应的细胞内环境的研究，比通常在典型试管中考虑的更忠实。拥挤非特异性地增强反应，导致总排除体积减少，例如溶液中大分子复合物的形成以及蛋白质的压缩或折叠。由于细胞内部，即使是最简单的原核系统，也包含各种具有空间和组成异质性的微环境，除了体积排斥之外，还必须考虑额外的特异性和非特异性相互作用的潜在影响，包括表面吸附和大分子分配。被认为是了解体内大分子反应性。这些挑战扩大了这个领域，并且已经进行了大量的实验和模拟研究，目的是至少部分地回答生物学相关的问题，即细胞中的大分子反应与试管中测量的反应有多大以及为何不同。 。 《化学评论》的这一虚拟专题旨在讨论分子拥挤的概念在过去二十年中如何演变，触及新的互补领域，以及拥挤研究中采用的新方法的引入，这些方法为分子拥挤研究提供了进一步的潜力。学习拥挤。在对该主题进行了历史介绍并分析了拥挤概念随时间的演变后，阿尔法诺等人。 (1) 介绍了该问题的其他评论中深入探讨的各个方面。除了蛋白质稳定性和蛋白质活性等众所周知的生物学主题外，本文还从胶体和聚合物物理学的角度探讨了拥挤的起源。他们还考虑了相变的拥挤效应，包括与蛋白质聚集、淀粉样蛋白形成和相分离生物分子凝聚相关的相变效应。他们还详细讨论了各种环境条件和使用不同拥挤器的价值。仔细选择合适的拥挤剂及其浓度对于在实验装置中模拟自然拥挤环境至关重要。拥挤研究的最终目标仍然是阐明其对生物功能的影响。为此，蒙特罗索等人。 (2)分析了大分子拥挤、相分离和物理化学稳态如何协调细菌细胞内组织和重要的细胞周期过程。他们首先讨论了影响细胞质结构的各种因素，包括拥挤和物理化学参数，如 pH、离子强度和能量状态，并将细菌中发现的超分子结构与真核细胞中的超分子结构进行了比较。 然后，他们分析了拥挤和相分离对细菌核组织的影响，以及染色体复制、分离和细菌细胞分裂过程的调节，以及这些过程的膜的影响。最后，他们讨论了相分离驱动的生物分子凝聚物如何与细菌适应性的调节相关。传统上，人们对生物大分子拥挤效应的关注几乎全部集中在蛋白质上。然而，正如 Zacco 等人 (3) 所讨论的，核酸所发挥的作用也至关重要，他们讨论了拥挤对 RNA 的影响。作者探讨了拥挤事件引起的颗粒或生物凝聚物中 RNA 序列、结构和化学修饰所发挥的关键作用。除了探索 RNA 在功能上重要的生理条件下发挥的作用之外，作者还研究了拥挤偏离其预期功能并导致病理后果的情况。他们还评估了用于破译 RNA 颗粒组成的方法，全面概述了用于表征它们的技术。总而言之，作者对 RNA 介导的凝聚体世界提供了多方面的理解。在当前关于分子拥挤的专题中，基于实验方法的贡献两侧是 Grassmann 等人的有影响力的论文。，（4）讨论了理论和计算方法，这些方法允许生物系统建模来指导和补充实验，从而可以显着推进细胞质拥挤环境中蛋白质-蛋白质相互作用的研究，甚至可能的预测。作者探讨了晶格模拟的统计力学、流体动力学相互作用、高粘度环境中的扩散过程以及基于分子动力学模拟的几种方法等主题。 Olgenblum 等人总是利用机会探索目前拥挤概念所涵盖的广泛主题。 (5)关注细胞可能面临的一种特定类型的应激：干燥应激。面对这种压力，许多生物体采取策略来维持其细胞成分的完整性。无定形玻璃介质提供了防止干燥的一般策略。他们回顾了这些策略和提出的分子机制，以解释低水合条件下玻璃体基质中的蛋白质保护。在探索拥挤所涵盖的主题多样性时，Peters 等人。 （六）解决极端条件下的拥挤问题。这组作者表示，如果我们要阐明各种外部条件下的生物功能，将环境考虑在内是多么重要，这一点正变得越来越清楚。许多生物系统在极端条件下茁壮成长，包括深海和海底地壳，并且可以利用拥挤的一些影响。近年来，人们利用各种生物物理技术研究了这些关系，包括中子和 X 射线散射、量热法、FTIR、紫外可见光谱和荧光光谱。 Peters 等人结合了极端条件下（例如温度、高静水压和高盐度）下生物分子的结构和构象动力学知识。强调了在环境背景下考虑结果的重要性。总体而言，新一代同步加速器辐射和更强大、更集中的计算方法等所带来的新技术和理论方法正在促进新的、引人注目的科学，希望为下一代研究人员提供信息并鼓励他们进一步探索可以设想新的方法来设计更忠实地模拟细胞内环境的实验。 Annalisa Pastore 是一位结构生物学家，在蛋白质结构测定方面拥有超过 35 年的经验，并对蛋白质稳定性、折叠和错误折叠有着浓厚的兴趣。在她获得硕士和博士学位之后。她在那不勒斯费德里科二世大学获得化学博士学位，之后在牛津大学和苏黎世联邦理工学院攻读博士后。 1988年，她搬到海德堡的欧洲分子生物学实验室（EMBL），并在那里建立了第一个生物核磁共振实验室。 1997 年至 2013 年，她在伦敦医学研究委员会工作，重点研究肌肉蛋白和与神经退行性变相关的蛋白质。 2013年至2021年在伦敦国王学院沃尔神经科学研究所工作。她目前隶属于伦敦国王学院、伦敦帝国学院和里雅斯特 Elettra Sincrotrone。 Germán Rivas Caballero 在 José Gonzalez-Rodríguez 实验室（西班牙马德里 CSIC 物理化学研究所）接受了博士培训。他获得了博士学位。 1989年获得西班牙马德里自治大学化学博士学位，随后在Allen Minton（NIH，1990-1992）和Jurgen Engel（1993，瑞士巴塞尔大学Biozentrum）实验室进行博士后培训。他自 1994 年以来一直在马德里 CSIC 的 CIB Margarita Salas 工作，自 1996 年起担任小组组长，自 2015 年起担任 CSIC 研究教授。他将自己的科学生涯致力于研究多蛋白系统，该系统的元素动态相互作用以组织功能性细胞参与重要流程的机器。在明顿实验室的博士后期间，他意识到局部微环境对生理（拥挤）环境中大分子关联的功能能量学的影响。他和他的同事开发了独特的生物物理方法来研究模拟细胞内部的拥挤条件下的蛋白质关联，使他们能够通过实验证明，由于拥挤而排除的体积效应可以显着影响蛋白质关联的模式和程度。在过去的 25 年里，Rivas 实验室一直在探索控制细菌分裂机制（分裂体）功能相互作用的生化机制，以在受控细胞样环境中采用自下而上的方法重建简化版本的分裂体。 Piero Andrea Temussi (PAT) 是一位结构生物学家，1963 年至 2010 年担任那不勒斯费德里科二世大学化学教授。在他的早期职业生涯中，他是 Paolo Corradini 和诺贝尔奖获得者 Giulio Natta 的合作者，后者引入了立体定向技术聚合。 PAT 是最早的两位科学家之一，与 Anna Laura Segre 一起，在 Herbert S 教授团队获得博士后经历后，在 70 年代早期在意大利引入了生物核磁共振 (NMR) 光谱技术。古托夫斯基在伊利诺伊大学厄巴纳-香槟分校。古托夫斯基是第一位将核磁共振方法应用于化学领域的科学家。 PAT还是以色列著名化学物理学家、魏茨曼科学研究所科学主任、以色列开放大学创始人Shneinor Lifson团队的访问科学家。 Lifson 以其一致力场方法而闻名，这是大分子 3D 计算机建模背后的主要理论之一。通过在意大利的工作，PAT 是阿斯巴甜和几种阿片类药物等生物活性相关肽的引人注目的研究的推动者和作者。他在知名期刊上发表了 200 多篇同行评审文章。 2015年至2021年，他担任伦敦国王学院（KCL）的客座教授。2019年，他成为欧洲科学院院士。他目前的科学兴趣范围从蛋白质折叠和稳定性到冷变性、拥挤和味道的分子理解。本文参考了其他 6 篇出版物。这篇文章尚未被其他出版物引用。本文参考了其他 6 篇出版物。