The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental ...challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
Down Syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and results in a spectrum of phenotypes including learning and memory deficits, and motor dysfunction. It has been hypothesized that ...an additional copy of a few Hsa21 dosage-sensitive genes causes these phenotypes, but this has been challenged by observations that aneuploidy can cause phenotypes by the mass action of large numbers of genes, with undetectable contributions from individual sequences. The motor abnormalities in DS are relatively understudied-the identity of causative dosage-sensitive genes and the mechanism underpinning the phenotypes are unknown. Using a panel of mouse strains with duplications of regions of mouse chromosomes orthologous to Hsa21 we show that increased dosage of small numbers of genes causes locomotor dysfunction and, moreover, that the Dyrk1a gene is required in three copies to cause the phenotype. Furthermore, we show for the first time a new DS phenotype: loss of motor neurons both in mouse models and, importantly, in humans with DS, that may contribute to locomotor dysfunction.
Integral membrane proteins (IMPs) and their interactions form the basis underlying many processes essential for life of all living organisms. Despite their pivotal importance, they are challenging to ...study due to the presence of a bilayer-embedded transmembrane domain requiring astute strategies to achieve solubilisation in aqueous environments. Here we present mass photometry as a novel technique to investigate IMPs and their interactions in a variety of membrane mimetic systems, uncovering the interactions underlying the bacterial outer membrane architecture. We then apply mass photometry to viral membrane glycoproteins, the HIV-1 envelope spike and SARS-CoV-2 spike, and their interactions with a soluble inhibiting protein and host cell receptor, respectively. In both cases, we describe how the oligomeric nature of the involved proteins facilitates intermolecular crosslinking, facilitating enhanced inhibition. We begin by establishing the applicability of mass photometry to measure a variety of membrane mimetic systems, including detergents, by determining the molecular weight of different IMPs in detergents, amphipols, lipid nanodiscs, and native nanodiscs. We further show the heterogeneity stemming from native membrane protein extraction with native nanodiscs, as well as the utility of mass photometry in predicting IMP functionality in different lipid nanodisc assemblies. We apply these findings to characterise the architecture and interactions between the IMP of the Gram-negative bacterial outer membrane, providing important insights into the outer membrane island formation and contributions to antibiotic resistance. Next, we focus on the interactions of the HIV-1 envelope spike glycoprotein with its natural inhibitor, banana lectin BanLec. Through careful mass photometry measurements, we determine the dynamics of this multivalent interaction, resulting in the formation of highly heterogeneous species through intermolecular crosslinking. This approach is then applied to investigate the mechanism behind SARS-CoV-2 infection. We measure the interactions between the viral spike and its human host cell receptor, ACE2, showing the importance of its dimeric nature for achieving its full neutralisation potential through spike crosslinking. This work introduced mass photometry as an important tool for membrane protein research with broad applications in protein-protein interactions as well as in characterisation of complex heterogeneous reactions important in viral infections.
Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, ...the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.
Mass Photometry of Membrane Proteins Olerinyova, Anna; Sonn-Segev, Adar; Gault, Joseph ...
Chem,
01/2021, Letnik:
7, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of ...mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers—in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.
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•We introduce a label-free, single molecule approach for membrane-protein characterization•Mass photometry quantifies membrane proteins in different membrane-mimetic systems•MP reveals carrier and protein heterogeneity•It helps distinguish different functional states of membrane proteins
Membrane proteins are some of the most important biological molecules, carrying out vital functions and being frequent drug targets. Yet, preferring lipid environments and so requiring solubilization, they are challenging to study. Here, we show that mass photometry can characterize the heterogeneity of membrane proteins and the carriers in which they are solubilized. It can also distinguish different functional states of membrane proteins. Our approach thus opens the door to more comprehensive studies of function, structure, and interaction of these critical proteins in their native membrane environment at the single-molecule level.
Membrane proteins are important in cell signaling and disease. They are also difficult to study as they require solubilization from lipids by membrane-mimetic systems. We show that mass photometry can facilitate the study of membrane proteins in various mimetic systems. With this method, we can distinguish different oligomeric and functional states of membrane proteins, opening the door for in vitro functional studies, structural characterization, and protein-protein interaction analysis of membrane proteins.
β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, ...through photoactivatable cross-linking into the
OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.
Quantitative mass imaging of single molecules Young, Gavin; Hundt, Nikolas; Cole, Daniel ...
Science (American Association for the Advancement of Science),
04/2018, Letnik:
360, Številka:
6387
Journal Article
Recenzirano
The cellular processes underpinning life are orchestrated by proteins and their interactions. Investigating the structural and dynamic heterogeneity of proteins, despite being key to their function, ...and their interaction with drugs, poses a fundamental challenge to existing analytical and structural methodologies. We use interferometric scattering microscopy to mass-image single biomolecules in solution with less than 2% mass error, up to 19-kDa resolution, and 1-kDa precision. We resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image proteins with associated lipids and sugars. These capabilities enable us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry provides spatially-resolved access to a broad range of biomolecular interactions, one molecule at a time.
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