Development and application of multicolor burst analysis spectroscopy

Shoup, Daniel; Roth, Andrew; Thapa, Rajan Kumar; Puchalla, Jason; Rye, Hays S.

doi:10.1016/j.bpj.2021.03.035

Cited by 3 publications

(21 citation statements)

References 49 publications

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“…For this experiment, two differently labeled RuBisCO samples were employed, one coupled to an Alexa488 dye (A488) and one coupled to an Alexa647 dye (A647), both at position 58. Because the spectral separation between these dyes is large, and they possess a very small Förster distance, the impact of FRET on the observed burst intensity distributions is negligible ( Shoup et al, 2021 ). Importantly, prior BAS studies demonstrate that changing the chemical identity of the exogenous fluorescent probe at the C58 labeling site has little impact on the population-resolved growth kinetics of the RuBisCO aggregates ( Shoup et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…Aggregates were formed with a 1:1 mixture of two, differently labeled RuBisCO monomers, one carrying a single Alexa488 dye and the other a single Alexa647 dye (pre-mixed). The Alexa488 and Alexa647 dyes display little FRET coupling under these conditions ( Shoup et al, 2021 ). Following formation of co-labeled aggregates (10 nM final total monomer), samples were supplemented with 1 µM DnaK, 2 µM DnaJ, 2 µM GrpE, and 200 nM ClpB, 2 mM ATP, and an ATP regeneration system.…”

Section: Resultsmentioning

confidence: 99%

“…Second, we employed a two-color version of BAS (MC-BAS) ( Shoup et al, 2021 ) to examine, in real time, the extent to which re-aggregation occurs during active disaggregation. For this experiment, two differently labeled RuBisCO samples were employed, one coupled to an Alexa488 dye (A488) and one coupled to an Alexa647 dye (A647), both at position 58.…”

Section: Resultsmentioning

confidence: 99%

“…MC-BAS employs both the observed raw burst amplitudes and associated burst ratios to reconstruct the size and stoichiometry distributions of co-labeled particles ( Shoup et al, 2021 ) When the RuBisCO-A488 and RuBisCO-A647 monomers are first co-aggregated, roughly a third of the total detected particles display strongly co-incident fluorescence bursts ( Figures 5A–C ). Following the addition of KJEB and ATP, the number of co-incident and non-coincident bursts decreases as the particles are disassembled ( Figure 5D ).…”

Section: Resultsmentioning

confidence: 99%

“…To circumvent these problems, we have applied a single particle technique known as Burst Analysis Spectroscopy (BAS) ( Puchalla et al, 2008 ) to directly study protein disaggregation by the E. coli bi-chaperone disaggregase system. BAS provides a free solution, minimally perturbative approach to measuring the population-resolved kinetics of aggregate disassembly, as well as way to study the real-time binding stoichiometries of molecular chaperones in distinct aggregate sub-populations [multi-color BAS or MC-BAS ( Shoup et al, 2021 )]. To minimize the impact of protein sequence variability, we establish conditions that permit the controlled formation of two different aggregate types from the same aggregation-prone protein, ribulose-1,5-carboxylase oxygenase (RuBisCO) from R. rubrum.…”

Section: Introductionmentioning

confidence: 99%

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The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones

Shoup

Roth

Puchalla

et al. 2022

Front. Mol. Biosci.

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Protein aggregation, or the uncontrolled self-assembly of partially folded proteins, is an ever-present danger for living organisms. Unimpeded, protein aggregation can result in severe cellular dysfunction and disease. A group of proteins known as molecular chaperones is responsible for dismantling protein aggregates. However, how protein aggregates are recognized and disassembled remains poorly understood. Here we employ a single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS), in combination with two structurally distinct aggregate types grown from the same starting protein, to examine the mechanism of chaperone-mediated protein disaggregation. Using the core bi-chaperone disaggregase system from Escherichia coli as a model, we demonstrate that, in contrast to prevailing models, the overall size of an aggregate particle has, at most, a minor influence on the progression of aggregate disassembly. Rather, we show that changes in internal structure, which have no observable impact on aggregate particle size or molecular chaperone binding, can dramatically limit the ability of the bi-chaperone system to take aggregates apart. In addition, these structural alterations progress with surprising speed, rendering aggregates resistant to disassembly within minutes. Thus, while protein aggregate structure is generally poorly defined and is often obscured by heterogeneous and complex particle distributions, it can have a determinative impact on the ability of cellular quality control systems to process protein aggregates.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones

Shoup

Roth

Puchalla

et al. 2022

Front. Mol. Biosci.

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Imaging single particle profiler to study nanoscale bioparticles using conventional confocal microscopy

Sych,

Görgens,

Steiner

et al. 2024

Preprint

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Single particle profiler is a unique methodology to study nanoscale bioparticles such as liposomes, lipid nanoparticles, extracellular vesicles and lipoproteins in single particle and high throughput manner. The original version requires the single photon counting modules for data acquisition. Here, we present imaging-based SPP (iSPP) which can be performed by imaging a spot over time in common imaging mode with photomultiplier tubes. We also provide a user-friendly software with graphical user interface to facilitate the application of this technique. We demonstrate that iSPP can be used to decipher lipid-protein interactions, membrane modifications by drugs and the heterogeneity of extracellular vesicles isolated from cells lines and urine of human donors.

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GTP‐stimulated membrane fission by the N‐BAR protein AMPH‐1

Kustigian

Xue

Gai

et al. 2022

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Membrane‐enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin‐like, Eps15‐homology domain (EHD) protein, RME‐1, functioning with the Bin/Amphiphysin/Rvs (N‐BAR) domain protein, AMPH‐1. Here we show, using a free‐solution single‐particle technique known as burst analysis spectroscopy (BAS), that AMPH‐1 alone creates small, tubular‐vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH‐1 and is slowed in the presence of RME‐1. Unexpectedly, AMPH‐1‐induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP‐stimulated membrane fission activity seen for AMPH‐1 is recapitulated by the heterodimeric N‐BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP‐stimulated membrane fission is a general property of this important class of N‐BAR proteins.

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Development and application of multicolor burst analysis spectroscopy

Cited by 3 publications

References 49 publications

The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones

The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones

Imaging single particle profiler to study nanoscale bioparticles using conventional confocal microscopy

GTP‐stimulated membrane fission by the N‐BAR protein AMPH‐1

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