Lerner, E. ; Ambrose, B. ; Barth, A. ; Birkedal, V. ; Blanchard, S. C. ; Borner, R. ; Cordes, T. ; Craggs, T. D. ; Ha, T. ; Haran, G. ; et al. The FRET-based structural dynamics challenge – community contributions to consistent and open science practices. arXiv הוגש, arXiv:2006.03091v1. Publisher's VersionAbstract
Single-molecule Förster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.
PDF icon smfret_position_paper_-_arxiv.pdf
Zaer, S. ; Drori, P. ; Lebendiker, M. ; Razvag, Y. ; Lerner, E. Abundant α-Synuclein compact dimers. הוגש. Publisher's VersionAbstract
α-Synuclein (αSyn) is an intrinsically disordered protein that forms oligomers and fibrils associated with Parkinson’s disease. As such, the mechanism of its oligomerization and its possible links to neurotoxicity have been the focus of many studies. Out of the numerous oligomer types, dimers are the smallest oligomers of aSyn that have been reported. As such, αSyn dimers serve as the earliest steps in the nucleation of αSyn oligomers and later fibrils. Therefore, it is important to characterize αSyn dimers. The identification of αSyn dimers in ensemble-averaged measurements without the use of chemical modifications have been difficult, due to their apparent low abundance. Using analytical anion exchange chromatography coupled to multi angle light scattering as well as to dynamic light scattering, we show that recombinant αSyn is in equilibrium between different types of monomers and compact dimers, and that both are abundant. Additionally, bulk Förster resonance energy transfer (FRET), fluorescence cross-correlation spectroscopy (FCCS) of FRET and pulsed-interleaved excitation single-molecule FRET (PIE smFRET) measurements of mixtures of donor- and acceptor-labeled αSyn. These measurements indicated a dimer dissociation constant of 1.75 μM. We concluded that αSyn dimers exist as abundant species in equilibrium with monomers only if produced to reach concentrations of hundreds of nanomolar or above.
Hagai, D. ; Lerner, E. Systematic Assessment of Burst Impurity in Confocal-Based Single-Molecule Fluorescence Detection Using Brownian Motion Simulations. Molecules 2019, 24, 2557. Publisher's VersionAbstract
Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements.
Segal, M. ; Ingargiola, A. ; Lerner, E. ; Chung, S. Y. ; White, J. A. ; Streets, A. M. ; Weiss, S. ; Michalet, X. High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins. Methods 2019. Publisher's VersionAbstract
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.
CHung, S. Y. ; Lerner, E. ; Jin, Y. ; Kim, S. ; Alhadid, Y. ; Grimaud, L. W. ; Zhang, I. X. ; Knobler, C. M. ; Gelbart, W. M. ; Weiss, S. The effect of macromolecular crowding on single-round transcription by Escherichia coli RNA polymerase. Nucleic Acid Research 2018. Publisher's VersionAbstract
Previous works have reported significant effects of macromolecular crowding on the structure and behavior of biomolecules. The crowded intracellular environment, in contrast to in vitro buffer solutions, likely imparts similar effects on biomolecules. The enzyme serving as the gatekeeper for the genome, RNA polymerase (RNAP), is among the most regulated enzymes. Although it was previously demonstrated that macromolecular crowding affects association of RNAP to DNA, not much is known about how crowding acts on late initiation and promoter clearance steps, which are considered to be the rate-determining steps for many promoters. Here, we demonstrate that macromolecular crowding enhances the rate of late initiation and promoter clearance using in vitro quenching-based single-molecule kinetics assays. Moreover, the enhancement’s dependence on crowder size notably deviates from predictions by the scaled-particle theory, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions could be affected by crowded conditions in the cellular milieu.
Lerner, E. ; Ingargiola, A. ; Weiss, S. Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example. J Chem Phys 2018, 148, 123315. bioRxiv pre-print versionAbstract
Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few “structural snapshots” along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique’s temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.
Ingargiola, A. ; Weiss, S. ; Lerner, E. Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions And Conformational Dynamics In Single-Molecule FRET. J Phys Chem B 2018, 122, 11598–11615. bioRxiv pre-print versionAbstract
Single-molecule Förster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Förster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances. Here we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distancedistribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.
Lerner, E. ; Cordes, T. ; Ingargiola, A. ; Alhadid, Y. ; Chung, S. ; Michalet, X. ; Weiss, S. Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer. Science 2018, 359. Publisher's VersionAbstract
Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.
Ingargiola, A. ; Peronio, P. ; Lerner, E. ; Gulinatti, A. ; Rech, I. ; Ghioni, M. ; Weiss, S. ; Michalet, X. 16-Ch Time-resolved Single-Molecule Spectroscopy Using Line Excitation. Proc SPIE Int Soc Opt Eng 2017, 10071. Publisher's VersionAbstract
Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.
Lerner, E. ; Ingargiola, A. ; Lee, J. J. ; Borukhov, S. ; Michalet, X. ; Weiss, S. Different types of pausing modes during transcription initiation. Transcription 2017, 8 242–253. Publisher's VersionAbstract
In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript.
Ingargiola, A. ; Lerner, E. ; Chung, S. ; Panzeri, F. ; Gulinatti, A. ; Rech, I. ; Ghioni, M. ; Weiss, S. ; Michalet, X. Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules. PLoS ONE 2017, 12, e0175766. Publisher's VersionAbstract
We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.
Alhadid, Y. ; Chung, S. ; Lerner, E. ; Taatjes, D. J. ; Borukhov, S. ; Weiss, S. Studying transcription initiation by RNA polymerase with diffusion-based single-molecule fluorescence. Protein Sci. 2017, 26, 1278–1290. Publisher's VersionAbstract
Over the past decade, fluorescence-based single-molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA-dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single-molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging.
Lerner, E. ; Chung, S. ; Allen, B. L. ; Wang, S. ; Lee, J. ; Lu, S. W. ; Grimaud, L. W. ; Ingargiola, A. ; Michalet, X. ; Alhadid, Y. ; et al. Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, E6562–E6571. Publisher's VersionAbstract
Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.
Ploetz, E. ; Lerner, E. ; Husada, F. ; Roelfs, M. ; Chung, S. ; Hohlbein, J. ; Weiss, S. ; Cordes, T. Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers. Sci Rep 2016, 6 33257. Publisher's VersionAbstract
Advanced 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 (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and 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 of E. coli RNA-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.
Ingargiola, A. ; Lerner, E. ; Chung, S. ; Weiss, S. ; Michalet, X. FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET. PLoS ONE 2016, 11, e0160716. Publisher's VersionAbstract
Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.
Lerner, E. ; Ploetz, E. ; Hohlbein, J. ; Cordes, T. ; Weiss, S. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET). J Phys Chem B 2016, 120, 6401–6410. Publisher's VersionAbstract
Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.
Orevi, T. ; Lerner, E. ; Rahamim, G. ; Amir, D. ; Haas, E. Ensemble and single-molecule detected time-resolved FRET methods in studies of protein conformations and dynamics. In Methods Mol. Biol. Methods Mol. Biol. 2014; Vol. 1076, pp. 113–169. Publisher's VersionAbstract
Most proteins are nanomachines that are selected to execute specific functions and therefore should have some degree of flexibility. The driving force that excites specific motions of domains and smaller chain elements is the thermal fluctuations of the solvent bath which are channeled to selected modes of motions by the structural constraints. Consequently characterization of the ensembles of conformers of proteins and their dynamics should be expressed in statistical terms, i.e., determination of probability distributions of the various conformers. This can be achieved by measurements of time-resolved dynamic non-radiative excitation energy transfer (trFRET) within ensembles of site specifically labeled protein molecules. Distributions of intramolecular segmental end-to-end distances and their fast fluctuations can be determined, and fast and slow conformational transitions within selected sections of the molecule can be monitored and analyzed. Both ensemble and single-molecule detection methods can be applied for data collection. In combination with synchronization methods, time-resolved FRET was also used for studies of fast conformational transitions, in particular the folding/unfolding transitions.
Lerner, E. ; Orevi, T. ; Ben Ishay, E. ; Amir, D. ; Haas, E. Kinetics of fast changing intramolecular distance distributions obtained by combined analysis of FRET efficiency kinetics and time-resolved FRET equilibrium measurements. Biophys. J. 2014, 106, 667–676. Publisher's VersionAbstract
Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRETefficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kineticstechnique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fastconformational transitions induced by common relaxation methods.
Nag, S. ; Sarkar, B. ; Chandrakesan, M. ; Abhyanakar, R. ; Bhowmik, D. ; Kombrabail, M. ; Dandekar, S. ; Lerner, E. ; Haas, E. ; Maiti, S. A folding transition underlies the emergence of membrane affinity in amyloid-β. Phys Chem Chem Phys 2013, 15, 19129–19133. Publisher's VersionAbstract
Small amyloid-β (Aβ) oligomers have much higher membrane affinity compared to the monomers, but the structural origin of this functional change is not understood. We show that as monomers assemble into small n-mers (n < 10), Aβ acquires a tertiary fold that is consistent with the mature fibrils. This is an early and defining transition for the aggregating peptide, and possibly underpins its altered bioactivity.
Lerner, E. ; Hilzenrat, G. ; Amir, D. ; Tauber, E. ; Garini, Y. ; Haas, E. Preparation of homogeneous samples of double-labelled protein suitable for single-molecule FRET measurements. Anal Bioanal Chem 2013, 405, 5983–5991. Publisher's VersionAbstract
Preparation of pure and homogenous site specifically single- and double-labelled biopolymers suitable for spectroscopic determination of structural characteristics is a major current challenge in biopolymers chemistry. In particular, proper analysis of single-molecule Förster resonance energy transfer measurements is based on the spectral characteristics of the probes. Heterogeneity of any of the probes may introduce errors in the analysis, and hence, care must be taken to avoid preparation of inhomogeneous labelled biopolymer samples. When we prepared samples of Escherichia coli adenylate kinase (AK) mutants labelled with either Atto 488 or Atto 647N, the products were spectrally inhomogeneous and the composition of the mixture changed gradually over time. We show here that the inhomogeneity was not a result of variation in the dye interaction with neighbouring side chains. Rather, the slow drift of the spectral characteristics of the probes was a characteristic of an irreversible chemical transformation probably due to the hydrolysis of the succinimide ring of the attached dye into its succinamic acid form. Overnight incubation of the labelled protein in mild basic solution accelerated the interconversion, yielding homogeneous labelled samples. Using this procedure, we obtained stable homogenous AK mutant labelled at residues 142 and 188.