Review
Chemistry, Multidisciplinary
Kiyohiko Kawai, Mamoru Fujitsuka, Atsushi Maruyama
Summary: Recent advances in fluorescence microscopy allow tracking of chemical reactions at the single-molecule level, minimizing sample amount needed for analysis and diagnosis, and enhancing efficiency. Single-molecule measurements show potential in ultralow-level biomarker detection, providing new perspectives in chemistry, physics, and life sciences.
ACCOUNTS OF CHEMICAL RESEARCH
(2021)
Review
Instruments & Instrumentation
Ziyu Yang, Haiqi Xu, Jiayu Wang, Wei Chen, Meiping Zhao
Summary: Fluorescence-based single-molecule techniques, such as FCS and smFRET, are powerful tools for analyzing the conformational dynamics and diversity of biological macromolecules, especially membrane proteins. This review covers the basic principles, labeling strategies, instrumental configurations, and data processing methods, as well as considerations for selecting fluorophores, labeling sites, experimental setups, and analysis methods for membrane protein dynamics analysis. Additionally, the remaining challenges and future development prospects in this field are briefly discussed.
APPLIED SPECTROSCOPY
(2021)
Review
Biochemistry & Molecular Biology
Samuel Naudi-Fabra, Martin Blackledge, Sigrid Milles
Summary: Single molecule fluorescence and nuclear magnetic resonance spectroscopy are powerful techniques for analyzing intrinsically disordered proteins. They provide complementary views to decipher the complex properties and interactions of IDPs, and have made significant contributions to our understanding of their molecular characteristics.
Article
Biochemical Research Methods
Jhullian J. Alston, Andrea Soranno, Alex S. Holehouse
Summary: Over the past two decades, intrinsically disordered proteins and protein regions have been recognized as essential drivers of cellular function, with singlemolecule fluorescence spectroscopy and molecular simulations providing insight into their behavior. When combined, these techniques offer complementary information that can help uncover complex molecular details.
Article
Chemistry, Physical
Oleg Opanasyuk, Anders Barth, Thomas-Otavio Peulen, Suren Felekyan, Stanislav Kalinin, Hugo Sanabria, Claus A. M. Seidel
Summary: This article introduces the importance of single-molecule Forster Resonance Energy Transfer (smFRET) experiments in resolving the structural dynamics of biomolecules. To overcome the challenges in these experiments, the authors propose a quantitative integrative analysis framework that accurately determines the number of states, their kinetic connectivity, transition rate constants, and species fractions in complex kinetic networks. They also provide a workflow for analysis and demonstrate the usefulness of this toolkit in dynamic structural biology.
JOURNAL OF CHEMICAL PHYSICS
(2022)
Article
Chemistry, Physical
Benjamin Vermeer, Sonja Schmid
Summary: Biomolecular systems rely on dynamic processes at the nanoscale, and detecting biomolecular nanodynamics is crucial for understanding the driving forces that control them. The technique of single-molecule fluorescence resonance energy transfer (smFRET) is limited by irreversible photobleaching, but the introduction of DyeCycling has the potential to break this limit and provide more information per single molecule. It has the ability to revolutionize time-resolved smFRET and uncover a wealth of biomolecular dynamics.
Article
Biochemistry & Molecular Biology
Yiming Zhang, Zongzhou Ji, Xin Wang, Yi Cao, Hai Pan
Summary: DNAzyme is a class of DNA molecules that can catalyze specific metal ions with high selectivity. Conformational changes in DNAzymes play an important role in catalysis, and these changes can be studied using the smFRET technique. In the case of a uranyl-specific DNAzyme 39E, conformational switches were observed to folded and extended states with the addition of Mg2+ and UO22+ respectively. The findings also showed that 39E can spontaneously undergo conformational changes between different configurations, with or without divalent metal ions.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Chemistry, Physical
Mirjam Kummerlin, Abhishek Mazumder, Achillefs N. Kapanidis
Summary: We present a general strategy to overcome photobleaching in single-molecule fluorescence measurements by replenishing fluorescent probes using transient binding of complementary DNA strands. This allows for near-continuous observation of single-molecule fluorescence for over an hour, much longer than typical photobleaching times. Our approach is adaptable to FRET and can be used to study the conformational dynamics of dynamic structures.
Article
Biology
Ashwin Balakrishnan, Katherina Hemmen, Susobhan Choudhury, Jan-Hagen Krohn, Kerstin Jansen, Mike Friedrich, Gerti Beliu, Markus Sauer, Martin J. Lohse, Katrin G. Heinze
Summary: The study explores the mobility of the beta 2-adrenergic receptor over a wide temporal range, from nanoseconds to seconds, using various fluorescence spectroscopy techniques. It reveals both translational and rotational mobility, which were previously hidden or overlooked, providing a comprehensive understanding of the receptor's dynamics.
COMMUNICATIONS BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Stefan Niekamp, Nico Stuurman, Nan Zhang, Ronald D. Vale
Summary: The study reveals that during motility, the motor protein dynein exhibits variability in the movement between its domains, with the AAA ring and MTBDs not always stepping simultaneously and taking differently sized steps. This variability results in a large number of unexpected conformational states of dynein during motility. The flexibility between major dynein domains is found to be critical for dynein motility.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Chemistry, Multidisciplinary
Alexandre Chappard, Craig Leighton, Rebecca S. S. Saleeb, Kiani Jeacock, Sarah R. R. Ball, Katie Morris, Owen Kantelberg, Ji-Eun Lee, Elsa Zacco, Annalisa Pastore, Margaret Sunde, David J. J. Clarke, Patrick Downey, Tilo Kunath, Mathew H. H. Horrocks
Summary: Protein misfolding and aggregation are common features of neurogenerative disorders. Single-molecule techniques have allowed the characterization of these protein aggregates, previously inaccessible with traditional methods. In this study, we employed a high affinity antibody labeled with orthogonal fluorophores, along with fast-flow microfluidics and single-molecule confocal microscopy, to specifically detect alpha-synuclein in biologically relevant samples.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2023)
Article
Chemistry, Multidisciplinary
Charanleen Kaur, Vishaldeep Kaur, Shikha Rai, Mridu Sharma, Tapasi Sen
Summary: Self-assembled plasmonic nanoantennas with extreme electromagnetic enhancement can be used to design ultrasensitive biosensors, which are highly beneficial for the effective detection of protein misfolding, early medical diagnosis, and prevention of neurodegenerative diseases.
Article
Optics
Raymundo Moya, Toru Kondo, Audrey C. Norris, Gabriela S. Schlau-Cohen
Summary: Single-molecule spectroscopy is widely used to study heterogeneity in behaviors on different timescales, with the recent emergence of single-molecule pump-probe spectroscopy for detecting heterogeneity on femtosecond and picosecond timescales. By developing a tunable apparatus, a bimodal distribution of energetic relaxation time constants was found for the fluorescent dye Atto647N, indicating dominance of specific processes within individual molecules and low heterogeneity. This spectrally-tunable single-molecule pump-probe spectroscopy has the potential to investigate heterogeneity in various biological and material systems.
Article
Multidisciplinary Sciences
Takanori Harashima, Shintaro Fujii, Yuki Jono, Tsuyoshi Terakawa, Noriyuki Kurita, Satoshi Kaneko, Manabu Kiguchi, Tomoaki Nishino
Summary: The electrical properties of DNA in the field of molecular electronics have been extensively studied, with a focus on the higher-order structures and design changes associated with single-molecule electronic devices. Researchers have developed a DNA zipper configuration to form a single-molecule junction, which exhibits high conductivity and an attractive self-restoring capability. This strategy provides a basis for novel designs of single-molecule junctions.
NATURE COMMUNICATIONS
(2021)
Article
Biochemistry & Molecular Biology
Olessya Yukhnovets, Henning Hofig, Nuno Bustorff, Alexandros Katranidis, Jorg Fitter
Summary: For single-molecule studies in solution, very low concentrations of dye-labelled molecules are needed to achieve single-molecule sensitivity. Specific demands on surface passivation are required to ensure stability of molecule concentration over a period of several hours. Diffusion rate and absolute molecule concentrations are important for controlling the number of molecules in the detection volume.
Article
Biochemical Research Methods
Alison Barbara Ross, Julian David Langer, Marko Jovanovic
Summary: Protein turnover, the continuous synthesis and degradation of proteins in cells, plays a crucial role in maintaining protein homeostasis and responding to stimuli. Studying the kinetics and dynamics of protein turnover can provide insights into how cells regulate essential processes such as growth, differentiation, and stress response.
MOLECULAR & CELLULAR PROTEOMICS
(2021)
Article
Biochemical Research Methods
Alexandros Lyratzakis, Giannis Valsamidis, Iliana Kanavaki, Antigoni Nikolaki, Fiona Rupprecht, Julian D. Langer, Georgios Tsiotis
Summary: This study focused on Pseudomonas sp. strain phDV1, a gram-negative bacterium capable of degrading aromatic compounds, and analyzed its proteome under different carbon sources. The research findings suggest that the carbon source influences the synthesis of enzymes related to the degradation of aromatic compounds and the production of polyhydroxyalkanoate (PHA).
Article
Hematology
Joanne C. Clark, Raluca A. I. Neagoe, Malou Zuidscherwoude, Deirdre M. Kavanagh, Alexandre Slater, Eleyna M. Martin, Mark Soave, David Stegner, Bernhard Nieswandt, Natalie S. Poulter, Johan Hummert, Dirk-Peter Herten, Michael G. Tomlinson, Stephen J. Hill, Steve P. Watson
Summary: The study found that GPVI is expressed as a mixture of monomers and dimers, and dimerization through the D2 domain is not critical for activation. Using FRET, it was shown that endogenous GPVI is expressed at least partially as a dimer on resting and activated platelet membranes.
THROMBOSIS AND HAEMOSTASIS
(2021)
Article
Biochemistry & Molecular Biology
Johan Hummert, Stanimir Asenov Tashev, Dirk-Peter Herten
Summary: Quantitative assessment of protein complexes, particularly in the context of cellular signaling, has become a pressing objective in cell biology. Recent advancements in single molecule fluorescence microscopy have led to different approaches for counting protein copy numbers in various cellular structures. Novel calibration protocols are needed to address challenges in molecular counting experiments.
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
(2021)
Review
Microbiology
Christian H. Ahrens, Joseph T. Wade, Matthew M. Champion, Julian D. Langer
Summary: Small proteins are abundant biomolecules, but they are often missed in genome annotations and difficult to identify using standard experimental approaches. Mass spectrometry has great potential for small protein discovery and characterization, but current methods have limitations. This review discusses the challenges and adjustments needed for small protein analysis using mass spectrometry, as well as future directions for improving their detection and characterization.
JOURNAL OF BACTERIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Michael J. Ziegler, Klaus Yserentant, Valentin Dunsing, Volker Middel, Antoni J. Gralak, Kaisa Pakari, Joern Bargstedt, Christoph Kern, Annett Petrich, Salvatore Chiantia, Uwe Strahle, Dirk-Peter Herten, Richard Wombacher
Summary: The agrochemical mandipropamid (Mandi) has been shown to be a highly efficient chemical inducer of protein proximity (CIP) in cell culture systems and living organisms, with high specificity and orthogonality. It is capable of rapid and efficient protein translocation and network manipulation.
NATURE CHEMICAL BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Maximilian Seidel, Anja Becker, Filipa Pereira, Jonathan J. M. Landry, Nayara Trevisan Doimo de Azevedo, Claudia M. Fusco, Eva Kaindl, Natalie Romanov, Janina Baumbach, Julian D. Langer, Erin M. Schuman, Kiran Raosaheb Patil, Gerhard Hummer, Vladimir Benes, Martin Beck
Summary: The authors investigate the structural motifs involved in co-translational assembly in nucleoporins and find that different paralogous assembly pathways have distinct co-translational assembly events. They identify several previously unknown co-translational interactions and demonstrate that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. These findings highlight the regulatory complexity of assembly pathways.
NATURE COMMUNICATIONS
(2022)
Article
Biology
Lea Kubitz, Sebastian Bitsch, Xiyan Zhao, Kerstin Schmitt, Lukas Deweid, Amelie Roehrig, Elisa Cappio Barazzone, Oliver Valerius, Harald Kolmar, Julien Bethune
Summary: Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has been established as a key technology for studying protein-protein interactions. To improve the efficiency and speed of PDB, various engineered enzymes have been developed, with ultraID being the smallest and most efficient enzyme to date. It allows for high-resolution investigation of protein interaction networks in different types of cells.
COMMUNICATIONS BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Fernando Muzzopappa, Johan Hummert, Michela Anfossi, Stanimir Asenov Tashev, Herten Dirk-Peter, Fabian Erdel
Summary: The article presents a new method to distinguish liquid-liquid phase separation (LLPS) from other mechanisms in cells, using well-controlled in vitro and in vivo experiments. The method, called MOCHA-FRAP, successfully identifies the interfacial barrier responsible for preferential internal mixing in biomolecular condensates. This research has important implications for understanding the formation and mechanisms of biomolecular condensates in living cells.
NATURE COMMUNICATIONS
(2022)
Article
Multidisciplinary Sciences
Eike Laube, Jakob Meier-Credo, Julian D. Langer, Werner Kuehlbrandt
Summary: The structure of mitochondrial complex I from Chaetomium thermophilum has been determined, revealing two distinct conformations and suggesting a universal mechanism of coupling electron transport to proton pumping.
Article
Multidisciplinary Sciences
Chao Sun, Kristina Desch, Belquis Nassim-Assir, Stefano L. Giandomenico, Paulina Nemcova, Julian D. Langer, Erin M. Schuman
Summary: The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Using super-resolution microscopy, researchers found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles, are distributed differently within rat cortical neurons. Surprisingly, an abundance of free 19S particles were discovered near synapses, which bind and remove lysine 63-ubiquitin, a non-proteasome-targeting ubiquitin linkage. Inhibition of the 19S deubiquitylase activity impacted synaptic transmission and reduced the availability of AMPA receptors in a proteasome-independent manner.
Article
Biology
Joanne C. Clark, Eleyna M. Martin, Luis A. Moran, Ying Di, Xueqing Wang, Malou Zuidscherwoude, Helena C. Brown, Deirdre M. Kavanagh, Johan Hummert, Johannes A. Eble, Bernhard Nieswandt, David Stegner, Alice Y. Pollitt, Dirk-Peter Herten, Michael G. Tomlinson, Angel Garcia, Steve P. Watson
Summary: Fluorescence correlation spectroscopy was used to investigate the clustering of CLEC-2 induced by crosslinked nanobody ligands. The results showed that the valency of the ligands and the expression levels of CLEC-2 were important factors in determining the activation of CLEC-2. Divalent ligands were found to be partial agonists while tetravalent ligands stimulated platelet aggregation.
COMMUNICATIONS BIOLOGY
(2023)
Article
Chemistry, Analytical
Jakob Meier-Credo, Benjamin Heiniger, Christian Schori, Fiona Rupprecht, Hartmut Michel, Christian H. Ahrens, Julian D. Langer
Summary: Small proteins have been overlooked in genetic and biochemical assays, but recent discoveries have highlighted their importance and potential as drug targets. The authors used an integrated approach to study small proteins in Pseudomonas stutzeri and identified known and novel proteins. This study demonstrates the power of the combined approach and its potential for studying small proteins in other prokaryotes.
ANALYTICAL CHEMISTRY
(2023)
Article
Biophysics
Felix Hild, Philipp Werther, Klaus Yserentant, Richard Wombacher, Dirk-Peter Herten
Summary: This study investigates the reaction sequence of trans-cyclooctene (TCO) with fluorogenic tetrazine dyes using a single-molecule fluorescence detection framework. The results provide insights into the transient intensity increase and decrease, and emphasize the need for robust benchmarking and alternative dienophiles for advanced fluorescence microscopy applications.
BIOPHYSICAL REPORTS
(2022)
Article
Biochemical Research Methods
Kristina Desch, Erin M. Schuman, Julian D. Langer
Summary: Cellular processes require precise control of protein abundance, localization, and activity. Protein phosphorylation is a core mechanism for specific regulation of proteins. This study presents a workflow using liquid chromatography-coupled mass spectrometry to monitor protein abundance and phosphorylation in primary cultured neurons. The protocol provides a comprehensive guide for detection and label-free quantification of phosphorylated and unmodified proteins, including primary cell culture, phosphoproteomic sample preparation, data processing, and evaluation.
