Raman Van Wee

Abstract:

DNA loop extrusion by cohesin has emerged as a critical pathway for chromosome organization. In vitro single-molecule experiments indicate that loop extrusion requires the assembly of a heteropentameric complex consisting of the SMC1/SMC3 heterodimer, STAG1, NIPBL, and the kleisin SCC1. The complexity of the complete extrusion machinery, consisting of multiple subunits, DNA binding sites, and ATPases poses substantial challenges for revealing the underlying biomolecular mechanism. As a result, a number of different models have been proposed, many of which do not agree on key mechanistic aspects, such as the details of DNA loading, holoenzyme assembly, or the consequences of ATP binding and hydrolysis. Here, we use mass photometry to comprehensively quantify all the key biomolecular interactions required for DNA loop extrusion. We find that STAG1 binds tightly to the trimeric complex formed by the SMC1/SMC3 heterodimer and SCC1, and together they weakly, but cooperatively, bind the DNA. Full-length NIPBL tightly binds DNA, acting as a DNA anchor during the mechanochemical loop extrusion cycle. Cohesin mutants incapable of head engagement, and those lacking DNA-binding domains in the ATPase heads show negligible differences in overall DNA-affinity, suggesting a minor role of these features for DNA binding. Instead, we find an ATP-modulated DNA binding site created by the interaction of STAG1 with SMC1/SMC3/SCC1, important for repeated grabbing and release of DNA critical to extrusion. Our results call for a careful reexamination of the proposed mechanisms and set energetic boundaries for future proposals.

Abstract:

Mass photometry (MP) is a technology for the mass measurement of biological macromolecules in solution. Its mass accuracy and resolution have transformed label-free optical detection into a quantitative measurement, enabling the identification of distinct species in a mixture and the characterization of their relative abundances. Its applicability to a variety of biomolecules, including polypeptides, nucleic acids, lipids, and sugars, coupled with the ability to quantify heterogeneity, interaction energies, and kinetics, has driven the rapid and widespread adoption of MP across the life sciences community. These applications have been largely orthogonal to those traditionally associated with microscopy, such as detection, imaging, and tracking, instead focusing on the constituents of biomolecular complexes and their change with time. Here, we present an overview of the origins of MP, its current applications, and future improvements that will further expand its scope.

Abstract:

Continuous biosensing provides real-time information about biochemical processes and holds great potential for health monitoring. Aptamers have emerged as promising alternatives over traditional biorecognition elements. However, the underlying aptamer-target binding interactions are often poorly understood. Here, we present a technique that can decode aptamer-protein binding interactions at the single-molecule level. We demonstrate that our single-molecule assay is able to decode the underlying binding kinetics of aptamers despite their similar binding affinity. Guided by computational simulations and validated with quartz crystal microbalance experiments, we show that the quantitative insights generated by this single-molecule technique enabled the rational understanding of biosensor performance (i.e., the sensitivity and limit of detection). This capability was demonstrated with thrombin as the analyte and the structurally similar aptamers HD1, RE31, and NU172 as the biorecognition elements. This work decodes aptamer-protein interactions with high temporal resolution, paving the way for the rational design of aptamer-based biosensors.

Abstract:

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.