Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biological processes and disease. Despite the importance of protein analysis, only a handful of techniques are available to determine protein sequences, and these methods face limitations, for example, requiring a sizable amount of sample. Single-molecule techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-molecule protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first experimental efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-molecule protein sequencing techniques.
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http://dx.doi.org/10.1038/s41565-018-0236-6 | DOI Listing |
J Am Chem Soc
March 2025
Université Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71 Avenue des Martyrs, Grenoble, Cedex 9 38044, France.
Green-to-red photoconvertible fluorescent proteins (PCFPs) of the EosFP family are commonly used in ensemble pulse-chase and single-molecule localization or tracking approaches. However, these fluorescent proteins exhibit highly complex photophysical behaviors. In the green-form, recent NMR experiments revealed that mEos4b and other PCFP variants exist in two different conformational states at thermal equilibrium, which limits their effective photoconversion efficiency.
View Article and Find Full Text PDFCell Rep Phys Sci
February 2025
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Multidrug efflux pumps confer not only antibiotic resistance to bacteria but also cell proliferation. In gram-negative bacteria, the ATP-binding cassette (ABC)-family transporter MacB, the adaptor protein MacA, and the outer membrane protein TolC form the MacA:MacB:TolC assembly to extrude antibiotics and virulence factors. Here, using quantitative single-molecule single-cell imaging, we uncover that, in cells, there is a large excess of MacB (and TolC) driving the limiting adaptor protein MacA mostly into the MacAB-TolC assembly.
View Article and Find Full Text PDFBiophys J
March 2025
Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, North Holland, The Netherlands; Lumicks B.V., Paalbergweg 3 1105 AG, Amsterdam, North Holland, The Netherlands. Electronic address:
The single-molecule biophysics community has delivered significant impacts to our understanding of fundamental biological processes, yet the field is also siloed and has fragmented data structures which impede data sharing and limit the ability to conduct comprehensive meta-analyses. To advance the field of optical tweezers in single-molecule biophysics, it is important that the field adopts open and collaborative data sharing that facilitate meta-analyses that combine diverse resources and supports more advanced analyses, akin to those seen in projects like the Protein Data Bank and the 1000 Genomes Project. Here, we assess the state of data findability, accessibility, interoperability, and reusability (the FAIR principles) within the single-molecule optical tweezers field.
View Article and Find Full Text PDFEMBO J
March 2025
Institute of Clinical Chemistry and Clinical Pharmacology, Biomedical Center II (BMZ II), Venusberg-Campus 1, University Hospital Bonn, University of Bonn, Bonn, 53127, Germany.
Widespread control of gene expression through translation has emerged as a key level of spatiotemporal regulation of protein expression. A prominent mechanism by which ribosomes can confer gene regulation is via internal ribosomal entry sites (IRESes), whose functions have however, remained difficult to rigorously characterize. Here we present a set of technologies in embryos and cells, including IRES-mediated translation of circular RNA (circRNA) reporters, single-molecule messenger (m)RNA isoform imaging, PacBio long-read sequencing, and isoform-sensitive mRNA quantification along polysome profiles as a new toolbox for understanding IRES regulation.
View Article and Find Full Text PDFACS Nano
March 2025
Faculty of Biomedical Engineering, Technion -IIT, Haifa 3200003, Israel.
Mitochondrial DNA (mtDNA) quantification is crucial in understanding mitochondrial dysfunction, which is linked to a variety of diseases, including cancer and neurodegenerative disorders. Traditional methods often rely on amplification-based techniques, which can introduce bias and lack the precision needed for clinical diagnostics. Solid-state nanopores, an emerging biosensing platform, have the advantage of offering single-molecule and label-free approaches by enabling the direct counting of DNA molecules without amplification.
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