Multiparameter Fluorescence Detection Research Articles

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50 μs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τD diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

Single-Molecule Fluorescence-Based Measurements of Conformational Dynamics of Calcium-Binding Protein Recoverin

Recoverin is a calcium-binding protein expressed primarily in the photoreceptor cells of the vertebrate retina, where it participates in the regulation of phototransduction and adaptation of the visual system to background light. Binding of calcium ions to recoverin induces large conformational changes, including the release of its post-translationally attached myristoyl group from a hydrophobic cavity of the protein into the solution. Previously, it was shown that the fluorophore Alexa647 site-specifically bound to a unique cysteine-39 of recoverin can be employed as a reporter of its Ca2+-dependent conformational transitions since these transitions affect fluorescence lifetime, and spectra of the dye. Here, we addressed a question, whether the Ca2+-dependent conformers of recoverin, previously resolved by NMR, can be recorded using single-molecule fluorescence microscopy. To this end, the samples of recombinant bovine recoverin site-specifically labeled at cysteine-39 with either Alexa647 or sulfo-Cy5 maleimide-functionalized fluorophores were prepared. Both forms of the protein exhibited Ca2+-induced conformational changes as revealed by steady-state spectrometry of intrinsic tryptophans fluorescence. Interestingly, two chemically similar cyanine dyes (Alexa647 and sulfo-Cy5) prodruced quite different effects on recoverin state and exhibited different photophysical responses to changes in its conformation. We performed single-molecule experiments with recoverin fluorescently labeled with Alexa647. Using a confocal setup with multiparameter fluorescence detection (MFD) we measured fluorescence lifetimes and anisotropies of bursts from individual recoverin molecules. Observation of individual molecules in solution is limited by their diffusion time (typically, 1-10 ms) - to track slower conformational changes we specifically immobilized recoverin using a biotin-streptavidin tether. Using a TIRF microscope we measured intensity traces from individual immobilized molecules. Our results show that conformational dynamics of recoverin can be measured at the single-molecule level using the protein site-specifically labeled at cysteine-39 with Alexa647. This work is supported by RFBR (№20-34-70034).

Resolving dynamics and function of transient states in single enzyme molecules

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.

Accurate FRET Measurements Resolving Distances and Dynamics in Biomolecules

Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of biomolecules in vitro and in live cells. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Förster Resonance Energy Transfer (FRET) to study the stoichiometry and structure of biomolecular systems. Before performing FRET measurements, one needs to know four calibration parameters, which depends on the photophysical properties of the used dyes and characteristics of optical components. We used a rational determination to estimate the calibration factors using DNA samples and compared this to a calibration free lifetime based analysis. This results in resolving individual γ-factors for every different set of dye pairs in different physical environments enabling an accurate monitoring of the operating dyes. Comparison and error estimation to the analysis with global parameters shows differences for specific dye pairs. Using this approach a network of FRET pairs in combination with integrative modeling is able to resolve the structure and dynamics of a biomolecule like the hGBP1. The shown extended network with more dye pairs results in a higher resolution of the structural model. To analyze the dynamic behavior of the biomolecule different several techniques like Multiparameter Fluorescence Detection (MFD), Photon Distribution Analysis (PDA) and filtered Fluorescence Correlation Spectroscopy (fFCS) are used. This workflow opens insights into several different dynamic proteins.

Interdomain Dynamics Underlie Function and Regulation of Postsynaptic Density Protein 95

Postsynaptic Density Protein 95 (PSD95) is a MAGUK-family scaffolding protein found in the excitatory postsynaptic density (PSD). PSD95 plays key roles in regulating synaptic organization and strength through interactions with neuroreceptors at the postsynaptic membrane. Consisting of 5 subdomains (3 PDZ domains, and SH3 domain, and a catalytically-inactive guanylate kinase (GuK) domain) connected by flexible polypeptide linkers, PSD95 further adopts a supertertiary structure in which the first two PDZ domains and the latter three domains partition into two supramodules (PDZ1-2 tandem and PSG supramodule). However, while the structures of the subdomains of PSD95 are well-studied, the underlying mechanisms governing PSD95 function and regulation are not entirely understood. A key characteristic of PSD95 is its structural dynamics. Dynamic intramolecular interactions between the five subdomains provide an attractive explanation for a degree of self-regulation of PSD95 that could extend to other PDZ-containing MAGUKs. In the present work we report a study of full-length PSD95, utilizing an integrative approach combining single-molecule FRET and multiparameter fluorescence detection (MFD) with discrete molecular dynamics (DMD) simulations and using biochemical methods for cross-validation. We analyzed 16 FRET variants to probe dynamics between pairwise combinations of PSD95 subdomains within and between the supramodules. Several key conclusions were drawn from this study: i) we corroborate previous results for the PDZ1-2 tandem identifying fast dynamics between two distinct conformations, now using full-length PSD95, ii) we characterize distinct dynamics within the supramodules and spanning the full PSD95, and iii) we identify three distinct, dynamically interacting structural conformations in the PSG supramodule via DMD and FRET screening. Our findings provide insights into how the interplay between structure and dynamics give rise to biomolecular function and regulation for PSD proteins.

Effects of Local and Global Dynamics on the Supertertiary Organization of Postsynaptic Density Protein 95

Postsynaptic density protein 95 (PSD-95) is a critical scaffolding protein in the excitatory postsynaptic density (PSD) with key functions in synaptic organization and regulation of synaptic strength. PSD-95 is a canonical multidomain scaffold protein comprised of three PDZ domains, an SH3 domain, and a guanylate “pseudokinase” (GuK) domain connected in series by flexible polypeptide linkers. The domains of PSD-95 show varying degrees of interaction and were suggested to partition into two independent supramodules. The first two PDZ domains display weak interactions but the third PDZ domain preferentially interacted with the SH3 and GuK domains. Despite a trove of structural studies, we do not understand how the interactions of PSD-95 with its diverse set of synaptic binding partners are regulated. PSD-95 exchanges between supertertiary configurations as part of its functional cycle. Interactions and posttranslational modifications can change the conformation with effects on function. This suggests that internal dynamics may regulate access to binding sites in PSD-95. In this work, we utilize a FRET network designed to probe all pairwise combinations of the five domains. We used multiparameter fluorescence detection and filtered fluorescence correlation spectroscopy to identify interdomain interactions and resolve dynamics from picoseconds to milliseconds. The labeling sites were on α-helices or surface loops. We distinguished locally rigid or flexible sites based on their relative contribution to FRET efficiencies and correlation amplitudes. Global analysis of the entire FRET network allowed comparison of the timescales associated with local and interdomain motions. Describing how local and interdomain dynamics drive the supertertiary organization of PSD-95 provides a basis for understanding how conformational changes regulate multidomain scaffold proteins.

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying atleast three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection oftranslocase mechanics and suggest models for possible PBD involvement during catalysis.

The E. coli Sec Reaction Pathway for Cellular Protein Sorting under a Single Molecule Loupe

Protein targeting and secretion is an essential biological process in all life forms. In E. coli, the Sec translocase machinery consists of a conserved protein-conducting channel (SecYEG), which associates with cytoplasmic partners such as SecA. Although busily studied, important mechanistic details on bacterial protein targeting and secretion are still lacking. Since protein conformation and dynamics seems to be key to the function of translocases, we therefore carried out quantitative studies on the dynamic conformation of SecA in vitro using single-pair Förster resonance energy transfer (sp-FRET). Here, single-molecule multi-parameter fluorescence detection (MFD) is a useful tool for sp-FRET, and when combined with nanosecond alternating donor/acceptor excitation, it provides a useful and robust readout of both donor and acceptor dye performance. Using FRET restrained structural modeling (FPS), which performs accessible volume calculations, we selected suitable residues for the used FRET pair (Atto488/Alexa647) on SecA. The different positions were mutated to cysteines for the creation of at least 30 different FRET-pairs, and optimal conditions for labeling with maleimide dyes were determined. Until now 7 FRET-pairs, which includes all the different domains, were selected for creating a dynamic 3D model of SecA. At the moment, we are investigating the effect of binding partners (e.g. pre-proteins such as PhoA) and co-factors (e.g. nucleotides, ADP and ATP) which will lead to very detailed SecA dynamics. We already showed that the addition of ADP results in a more stable SecA conformations. Later on we will me move to an in vitro translocation assay to study the mechanism of substrate docking and –translocation on SecYEG, based on functional existing assays which are compatible with single-molecule experiments.

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination.

A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A Multiscalar Framework describes Fluorescence and FRET of Fluctuating Molecular Species and Resolves Kinetic Networks

A combination of multi parameter fluorescence detection (MFD) with structure based fluorescence models is presented, to capture and describe fluorescence and FRET between a donor (D) and acceptor (A) dye of fluctuating macromolecules over more than five orders of magnitude from picoseconds to seconds with Angstrom resolution. The presented top down approach combines molecular models with established fluorescence techniques such as time correlated single photon counting, burst integrated fluorescence lifetime analysis, filtered fluorescence correlation spectroscopy (fFCS), and photon distribution analysis in a joined framework and thus facilitates the analysis and interpretation of fluorescence experiments. Fluorescence and FRET on the picoseconds to nanosecond regime is described by combining atomistic models with a coarse grained representation of the dyes. Their conformational space is quantified by coarse grained accessible volume simulations while Brownian dynamics simulations capture transient effects of FRET and fluorescence quenching. Assuming dynamic quenching and FRET are decoupled, the first two modes of the DA-distance distribution are determined for single molecules to conveniently reveal macromolecular kinetics on the milli- and sub-millisecond kinetics by MFD histograms. Structural models are projected to yield parametric equations of single-molecule observables. This serves as a visual guide to analyse MFD- histograms. Numeric integration of the chemical master equation analyses MFD-histograms and quantifies in combination with fFCS kinetic networks of dynamically exchanging macromolecular conformations in the sub-microsecond to millisecond regime. A joint analysis of multiple fluorescence decays by structure based patterns resolved chemical equilibria in live cell. In future, such holistic approaches may exploit the information contained in the fluorescence signal and connect dynamic molecular structural models with kinetic and equilibrium networks of biomolecules to picture molecular machines in living cells.

Fluorescence Evidences for Non-Homogeneity and Residual Structure of Denatured States

About 30 % of human proteins do not fold into a stable 3D arrangement of secondary structure elements, but stay predominantly unfolded -similar to proteins under highly denaturing conditions. These proteins are involved in many cell signaling processes. Their characterization poses a great challenge for current experimental methods as they consist of an ensemble of rapidly interconverting conformations. Intense debate exists on the possibility that they show, to certain extent, residual structure, which might facilitate folding or enhance ligand binding. To study the unfolded state conformational heterogeneity using Forster resonance energy transfer (FRET), we used the lysozyme from the phage T4 (T4L) in denaturing conditions as a model system. We built an elastic network model that spans T4L's topology in order to evaluate local and global conformational changes by combining ensemble (ensemble time-resolved fluorescence lifetime and anisotropy) and single-molecule spectroscopic (multiparameter fluorescence detection, photon distribution analysis, (filtered) fluorescence correlation spectroscopy) methods. Through extensive comparison of models, we identified regions with apparent residual structure under highly denaturing conditions, which might serve as folding nuclei; and additionally we showed that chemically denatured T4L is not a random coil as previously thought. By using obtained distance restraints we determined that denatured T4L shows a native-like mean structure, albeit larger in size compared to the native state. We demonstrate here the necessity of careful data interpretation, but also the potential of a multidimensional approach to characterize an ensemble of states, which can be applied generally to unstructured or denatured proteins.

Structural Dynamics of Single Metabotropic Glutamate Receptors in Solution

Metabotropic glutamate receptors (mGluRs) are multidomain proteins belonging to class C G-protein coupled receptors (GPCR). There are essential in controlling synaptic transmission, and as such are important drug targets for the treatment of several disorders including pain, Parkinson's disease, schizophrenia etc.We recently introduced the use of single molecule FRET combined with Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to investigate the allosteric transitions associated with mGluR activation. We demonstrated that the isolated ligand binding (VFT) domain oscillates between a resting and an active state in a time range of 50-100 µs∗. Here, we extend these investigation to the full length receptor solubilized in detergent and reconstituted in liposomes. Our result confirm the general mechanism observed for the VFT domain, and decipher the role of the transmembrane domain, that slightly slows down the receptor dynamics, while stabilizing its active state solely in the presence of a full agonist.∗Olofsson et al., Nature Comm., 2015

Accurate Determination of the RNA Three-Way Junctions Via Single-Molecule High-Precision Fret Measurements

In nature RNA helices are often linked via various junctions and bulges that determine overall 3D structure which are used as building blocks and functional components in nanotechnology applications. Hence visualization of RNA conformations is crucial for understanding their biological functions. Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling can be used to determine the structure of these junctions. Multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting (eTCSPC) measurements were applied to perform FRET study on systematic series of 6 RNA three- way-junctions (3WJs) derived from the hairpin ribozyme. Bulge and sequence variations were considered as dominant factors influencing junction, two of which have bulge (two and five unpaired nucleotides) in the junction region. We have generated a database of overall 252 different RNA 3WJs labeled with Alexa488 and Cy5 fluorescent dyes. The analysis toolkit included probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation. We detected only one predominant conformer for RNAs 3WJ. Furthermore we report that bulges in the junction determine orientation and rotation of helices, inducing coaxial stacking. We provide a geometrical description of the junction geometry and our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality. As the accuracy of FRET restrained modeling depends on the correct description of the dye behavior, we characterized the position dependent fluorescent properties of the coupled dyes and statistically analyzed the systematic deviations of the calculated FRET distances from the model generated distances. This allowed us to identify the problematic labeling positions. Thus we could improve the accuracy of the FRET method by careful selection of labeling sites.

Submillisecond Dynamics of the NMDA Receptor

Observation of single molecules has impacted the way we look at biomolecular machines. Particularly, because it gave us a tool to understand how biomachines move as they carry out specific functions. Single molecule fluorescence experiments, done in surface immobilized conditions or freely diffusing, take advantage of the fact that fluorescence occurs in the nanosecond timescale to map a wide range of biologically relevant dynamics, covering over 10 orders of magnitude in time without gaps. Single molecule Forster Resonance Energy Transfer (smFRET) experiments have evolved to the point where it is possible to work with complex systems including membrane proteins. However, many challenges have limited the impact on the field, mostly because there is the common conception that the obtained structural information is very limited. In addition, multiple experimental artifacts can complicate the interpretation of data. However, recent advances in smFRET experiments, particularly using Multiparameter Fluorescence Detection (MFD), have improved its accuracy and precision, to the point that it is possible to overcome this mindset. As an example, we use MFD to identify low populated conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics in the sub millisecond regime and identify how conformational dynamics sheds light into the mechanism of partial agonism in the GluN1 subunit of the NMDA receptor.

Multi-Parameter Fluorescence Spectroscopy and Imaging for Quantitative FRET Measurements

Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of biomolecules in vitro and in live cells. However, often our view of molecular function is still formed, to a significant extent, by traditional structure determination showing many detailed static snapshots of biomolecular structures. Recent fluorescence experiments added a dynamic perspective by showing the heterogeneity and flexibility of molecular structures, visualizing transiently populated conformational states and identifying exchange pathways. This dynamic personality is an essential aspect for understanding their function. Following the ideas of Theodor Forster and Gregorio Weber, I introduce multi-parameter fluorescence detection (MFD) [1] and multi-parameter fluorescence image spectroscopy (MFIS) [2]. These techniques register all eight characteristic fluorescence parameters in a single measurement so that we can gain maximum resolution of specific fluorescence information on the biomolecule. The five intrinsic fluorescence properties of a fluorophore such as spectral properties in absorption and in fluorescence, brightness and quantum yield, lifetime and anisotropy report on its local environment. Three fluorescence parameters are connected to the system. The application of fluctuation spectroscopy allows us to resolve system properties such as diffusional properties and kinetic networks. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Forster Resonance Energy Transfer (FRET) to study the stoichiometry and structure of biomolecular systems. Taking the large GTPase human Guanylate binding protein 1 (hGBP1) as example, I will present the structural and dynamical characterization of all intermediates during hGBP1 oligomerization by high-precision FRET in vitro and in live cells [4]. [1] Widengren et al.; Anal. Chem. 78, 2039-2050 (2006). [2] Weidtkamp-Peters et al.; Photochem. Photobiol. Sci., 8, 470-480 (2009). [3] Kalinin et al.; Nat. Methods 9, 1218-1225 (2012). [4] Kravets et. al.; eLife 5, e11479 (2016).

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Resolving the Heterogeneity of the Ensemble of Unfolded States by a Combination of Fluorescence Spectroscopic Methods

Intense debate exists on the possibility that unfolded proteins show, to certain extent, residual secondary structure. Transient structure formation might facilitate folding (nucleation site) and/ or enhance binding to ligands. Some proteins such as those categorized as intrinsically disordered proteins take advantage of these features. Here, we used the model enzyme lysozyme from the phage T4 (T4L), a simple two subdomain protein, which already exists in an equilibrium of at least three conformations under native conditions. We created a set of 24 double mutants of the cysteine-free pseudo-wild type by inserting an unnatural amino acid and a cysteine mutation and site-specifically labeled them using orthogonal chemistry with a Forster resonance energy transfer (FRET) dye pair. Our set of variants allows us to build up a network of distances spanning the enzyme in order to monitor the ensemble of conformations. The behavior of the protein under highly denaturing conditions was observed by a combination of ensemble (ensemble time-resolved fluorescence lifetime and anisotropy) and single-molecule spectroscopic (multiparameter fluorescence detection, photon distribution analysis, (filtered) fluorescence correlation spectroscopy) methods. Our network covered all possible directions over the whole protein allowing us to map specifically the local motions (dye mobility) and global changes. We identified regions with residual structure which exist even under highly denaturing conditions. Additionally, the combination of ensemble and single-molecule methods allows us to resolve the full heterogeneity of the proteins’ denatured conformations.

Studying the Function of BAP in the Nucleotide Cycle of BIP by spFRET using MFD-PIE

Heat shock proteins, like Hsp70 and Hsp90, act as chaperones, a class of proteins that assist nascent proteins in reaching their final functional structure in the appropriate location within the cell. Understanding the underlying mechanisms of chaperone-assisted protein folding is important for the development of treatment for various disorders such as Alzheimer or Parkinson's disease. BiP, the Hsp70 localized in the endoplasmic reticulum (ER), consists of two domains that are connected by a short linker, the nucleotide binding domain (NBD) and the substrate binding domain (SBD). The SBD contains a flexible, alpha helical lid that can open or close the binding pocket of the SBD. When a client protein enters the ER, its charged regions are initially protected by BiP to avoid non-specific interactions. One of the regulating factors in this process is the BiP associated protein, BAP, a nucleotide exchange factor (NEF) of BiP. BAP controls the binding and release of nascent proteins from BiP by accelerating the ATP/ADP exchange. In order to gain insights into the function of BAP, we employed solution based single-pair Forster Resonance Energy Transfer (spFRET) experiments using a Multiparameter Fluorescence Detection (MFD) setup combined with Pulsed Interleaved Excitation (PIE). BiP was fluorescently labeled with the FRET pair Atto532 and Atto647. Our results suggest that the N-terminal domain of BAP interacts with the lid of BiP and pushing the two BiP domains apart from each other. Furthermore we could detect how BAP functioning as a NEF. Without BAP, the lid opens twice during one nucleotide cycle, first upon binding of ATP and secondly when phosphate is released. By adding BAP, the lid stays in its open conformation during hydrolysis and the release of phosphate and ADP is enhanced.

Structural Dynamics of the Full-Length Metabotropic Glutamate Receptors by Single-Molecule FRET

G protein-coupled receptors (GPCRs) constitute the most abundant protein family in mammalian genome. Indeed, GPCRs are the target for about 30% of the drugs on the market. In human particularly, there are 22 genes encoding class C GPCRs, which consists of GABAb receptor, calcium sensing receptor, retinoic acid-inducible receptors (orphan), taste receptors, metabotropic glutamate receptors, and a few additional orphan receptors. Metabotropic glutamate receptors (mGluRs) are involved in controlling synaptic transmission, and involved in various CNS disorders including pain, Parkinson's disease, schizophrenia etc... They are naturally homodimers, and each protomer consists of a heptahelical transmembrane domain linked to a bilobate Venus flytrap domain (VFT) by a Cystein-rich domain. Understanding the conformational changes of mGluRs is essential to decipher the allosteric transition associated with their activation. Crystallographic studies suggested that in the inactive and active states the second lobes of the VFTs are distant and close, respectively. However certain ambiguities and discrepancies about these two states have been observed by X-ray crystallography. For the first time in GPCR activation mechanism studies, we proposed structural dynamics investigations by single-molecule FRET (sm-FRET) in solution. Our results show that the isolated mGluR VFTs oscillate between the resting and active states in a time range of 50-100μs (Olofsson et al. Nat. Comm. 2014). Following the success of the powerful sm-FRET methodology on isolated VFTs, we employ here Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to study the full-length receptors in order to gain additional insights into mGluR activation. Our current results confirm the structural dynamics obtained by the mGluRs VFT, and suggest a stabilizing role of the transmembrane domain. Further studies on allosteric modulation and G protein effect are on-going.