scholarly journals Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nadia Füllbrunn ◽  
Zehao Li ◽  
Lara Jorde ◽  
Christian P Richter ◽  
Rainer Kurre ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Effector Proteins ◽  
Axial Orientation ◽  
Biological Processes ◽  
Time Resolved ◽  
Macromolecular Complexes ◽  
Open Conformation

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a ‘closed’ and an ‘open’ conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

scholarly journals Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2057 ◽  
Author(s):  
Nathalie Meiser ◽  
Christin Fuks ◽  
Martin Hengesbach
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Protein Complexes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Telomere Maintenance ◽  
Förster Resonance Energy Transfer ◽  
Rna Protein Complexes ◽  
Förster Resonance

RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.

scholarly journals Rapid extraction and kinetic analysis of protein complexes from single cells

2021 ◽  
Author(s):  
Sena Sarıkaya ◽  
Daniel J Dickinson
Keyword(s):  
Single Molecule ◽  
Protein Complex ◽  
Cell Biology ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Single Cells ◽  
Cell Lysis ◽  
Cellular Protein ◽  
In Vitro Assays

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays, while being applicable to native protein complexes isolated from single cells in vivo.

scholarly journals Energy Transfer in Reconstituted Peridinin-Chlorophyll-Protein Complexes: Ensemble and Single-Molecule Spectroscopy Studies

2007 ◽  
Vol 93 (9) ◽  
pp. 3249-3258 ◽  
Author(s):  
Sebastian Mackowski ◽  
Stephan Wörmke ◽  
Tatas H.P. Brotosudarmo ◽  
Christophe Jung ◽  
Roger G. Hiller ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Protein Complexes ◽  
Single Molecule Spectroscopy ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein ◽  
Spectroscopy Studies

scholarly journals A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

2011 ◽  
Vol 16 (3) ◽  
pp. 356-362 ◽  
Author(s):  
Eija Martikkala ◽  
Anita Rozwandowicz-Jansen ◽  
Pekka Hänninen ◽  
Ulla Petäjä-Repo ◽  
Harri Härmä
Keyword(s):  
Energy Transfer ◽  
Adenylyl Cyclase ◽  
High Throughput ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Gpcr Activation ◽  
Label Time

G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.

Methods to analyse composition and dynamics of macromolecular complexes

2013 ◽  
Vol 41 (5) ◽  
pp. 1235-1241 ◽  
Author(s):  
Heinrich Heide ◽  
Ilka Wittig
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Biosynthesis ◽  
Biological Processes ◽  
Protein Protein Interactions ◽  
Macromolecular Complexes ◽  
Cellular Processes ◽  
Recent Developments ◽  
Dna Replication And Repair ◽  
Health And Disease

Macromolecular complexes are involved in a broad spectrum of cellular processes including protein biosynthesis, protein secretion and degradation, metabolism, DNA replication and repair, and signal transduction along with other important biological processes. The analysis of protein complexes in health and disease is important to gain insights into cellular physiology and pathophysiology. In the last few decades, research has focused on the identification and the dynamics of macromolecular complexes. Several techniques have been developed to isolate native protein complexes from cells and tissues to allow further characterization by microscopic and proteomic analysis. In the present paper, we provide a brief overview of proteomic methods that can be used to identify protein–protein interactions, focusing on recent developments to study the entire complexome of a biological sample.

scholarly journals Single-Molecule Techniques to Study Chromatin

2021 ◽  
Vol 9 ◽  
Author(s):  
Anna Chanou ◽  
Stephan Hamperl
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Protein Complexes ◽  
Population Based ◽  
Biological Processes ◽  
Transcription Analysis ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Eukaryotic Chromatin ◽  
Genomic Regions

Besides the basic organization in nucleosome core particles (NCPs), eukaryotic chromatin is further packed through interactions with numerous protein complexes including transcription factors, chromatin remodeling and modifying enzymes. This nucleoprotein complex provides the template for many important biological processes, such as DNA replication, transcription, and DNA repair. Thus, to understand the molecular basis of these DNA transactions, it is critical to define individual changes of the chromatin structure at precise genomic regions where these machineries assemble and drive biological reactions. Single-molecule approaches provide the only possible solution to overcome the heterogenous nature of chromatin and monitor the behavior of individual chromatin transactions in real-time. In this review, we will give an overview of currently available single-molecule methods to obtain mechanistic insights into nucleosome positioning, histone modifications and DNA replication and transcription analysis—previously unattainable with population-based assays.

scholarly journals Fluorescence resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

2016 ◽  
Author(s):  
Evelyn Ploetz ◽  
Eitan Lerner ◽  
Florence Husada ◽  
Martin Roelfs ◽  
SangYoon Chung ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Fluorescence Enhancement ◽  
Protein Complexes ◽  
Fundamental Problem ◽  
Resonance Energy Transfer ◽  
Resonance Energy

ABSTRACTAdvanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (viaPIFE) and energy transfer efficiency (viaFRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching ofE. coliRNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

scholarly journals Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept

2021 ◽  
Vol 8 ◽  
Author(s):  
Taryn M. Kay ◽  
Cody P. Aplin ◽  
Rowan Simonet ◽  
Julie Beenken ◽  
Robert C. Miller ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ensemble Averaging ◽  
Time Resolved ◽  
Single Molecule Level ◽  
Fluorescence Measurements ◽  
Averaging Time ◽  
Molecular Brightness

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.

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