Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 µm2/s), slow (∼0.04 µm2/s), and fast (∼0.14 µm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

Local Disordered Region Sampling (LDRS) for ensemble modeling of proteins with experimentally undetermined or low confidence prediction segments.

SUMMARY: The Local Disordered Region Sampling (LDRS, pronounced loaders) tool is a new module developed for IDPConformerGenerator, a previously validated approach to model intrinsically disordered proteins (IDPs). The IDPConformerGenerator LDRS module provides a method for generating all-atom conformations of intrinsically disordered protein regions at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures. Requiring only a pre-existing PDB or mmCIF formatted structural template of the protein with missing coordinates or with predicted confidence scores and its full-length primary sequence, LDRS will automatically generate physically meaningful conformational ensembles of the missing flexible regions to complete the full-length protein. The capabilities of the LDRS tool of IDPConformerGenerator include modeling phosphorylation sites using enhanced Monte Carlo-Side Chain Entropy, transmembrane proteins within an all-atom bilayer, and multi-chain complexes. The modeling capacity of LDRS capitalizes on the modularity, the ability to be used as a library and via command-line, and the computational speed of the IDPConformerGenerator platform. AVAILABILITY AND IMPLEMENTATION: The LDRS module is part of the IDPConformerGenerator modeling suite, which can be downloaded from GitHub at https://github.com/julie-forman-kay-lab/IDPConformerGenerator. IDPConformerGenerator is written in Python3 and works on Linux, Microsoft Windows, and Mac OS versions that support DSSP. Users can utilize LDRS's Python API for scripting the same way they can use any part of IDPConformerGenerator's API, by importing functions from the "idpconfgen.ldrs_helper" library. Otherwise, LDRS can be used as a command line interface application within IDPConformerGenerator. Full documentation is available within the command-line interface as well as on IDPConformerGenerator's official documentation pages (https://idpconformergenerator.readthedocs.io/en/latest/).

Local Disordered Region Sampling (LDRS) for Ensemble Modeling of Proteins with Experimentally Undetermined or Low Confidence Prediction Segments.

The Local Disordered Region Sampling (LDRS, pronounced loaders) tool, developed for the IDPConformerGenerator platform (Teixeira et al. 2022), provides a method for generating all-atom conformations of intrinsically disordered regions (IDRs) at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures. Requiring only a pre-existing PDB structure of the protein with missing coordinates or with predicted confidence scores and its full-length primary sequence, LDRS will automatically generate physically meaningful conformational ensembles of the missing flexible regions to complete the full-length protein. The capabilities of the LDRS tool of IDPConformerGenerator include modeling phosphorylation sites using enhanced Monte Carlo Side Chain Entropy (MC-SCE) (Bhowmick and Head-Gordon 2015), transmembrane proteins within an all-atom bilayer, and multi-chain complexes. The modeling capacity of LDRS capitalizes on the modularity, ability to be used as a library and via command-line, and computational speed of the IDPConformerGenerator platform.

Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modeling and Clustering.

The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces the level of eIF4E binding, in part by stabilizing a binding-incompatible folded domain. Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and 5-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively). We then applied an integrative Bayesian approach to obtain NP and 5P conformational ensembles that agree with experimental data from nuclear magnetic resonance, small-angle X-ray scattering, and single-molecule Förster resonance energy transfer (smFRET). For the NP state, inter-residue distance scaling and 2D maps revealed the role of charge segregation and pi interactions in driving contacts between distal regions of the chain (∼70 residues apart). The 5P ensemble shows prominent contacts of the N-IDR region with the two phosphosites in the folded domain, pT37 and pT46, and, to a lesser extent, delocalized interactions with the C-IDR region. Agglomerative hierarchical clustering led to partitioning of each of the two ensembles into four clusters with different global dimensions and contact maps. This helped delineate an NP cluster that, based on our smFRET data, is compatible with the eIF4E-bound state. 5P clusters were differentiated by interactions of C-IDR with the folded domain and of the N-IDR with the two phosphosites in the folded domain. Our study provides both a better visualization of fundamental structural poses of 4E-BP2 and a set of falsifiable insights on intrachain interactions that bias folding and binding of this protein.

Integrative Conformational Ensembles of Sic1 Using Different Initial Pools and Optimization Methods.

Intrinsically disordered proteins play key roles in regulatory protein interactions, but their detailed structural characterization remains challenging. Here we calculate and compare conformational ensembles for the disordered protein Sic1 from yeast, starting from initial ensembles that were generated either by statistical sampling of the conformational landscape, or by molecular dynamics simulations. Two popular, yet contrasting optimization methods were used, ENSEMBLE and Bayesian Maximum Entropy, to achieve agreement with experimental data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule Förster resonance energy transfer. The comparative analysis of the optimized ensembles, including secondary structure propensity, inter-residue contact maps, and the distributions of hydrogen bond and pi interactions, revealed the importance of the physics-based generation of initial ensembles. The analysis also provides insights into designing new experiments that report on the least restrained features among the optimized ensembles. Overall, differences between ensembles optimized from different priors were greater than when using the same prior with different optimization methods. Generating increasingly accurate, reliable and experimentally validated ensembles for disordered proteins is an important step towards a mechanistic understanding of their biological function and involvement in various diseases.

Single-molecule counting applied to the study of GPCR oligomerization.

Single-molecule counting techniques enable a precise determination of the intracellular abundance and stoichiometry of proteins and macromolecular complexes. These details are often challenging to quantitatively assess yet are essential for our understanding of cellular function. Consider G-protein-coupled receptors-an expansive class of transmembrane signaling proteins that participate in many vital physiological functions making them a popular target for drug development. While early evidence for the role of oligomerization in receptor signaling came from ensemble biochemical and biophysical assays, innovations in single-molecule measurements are now driving a paradigm shift in our understanding of its relevance. Here, we review recent developments in single-molecule counting with a focus on photobleaching step counting and the emerging technique of quantitative single-molecule localization microscopy-with a particular emphasis on the potential for these techniques to advance our understanding of the role of oligomerization in G-protein-coupled receptor signaling.

Multisite phosphorylation and binding alter conformational dynamics of the 4E-BP2 protein.

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamic characterization of their ensembles remain challenging, both in isolation and when they form dynamic "fuzzy" complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Förster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a non-uniform segmental flexibility around six different labeling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-to-microsecond timescales. Upon hyperphosphorylation, which induces folding of ∼40 residues in 4E-BP2, the quenching rates decreased at most labeling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step toward a mechanistic understanding of this important IDP via integrative modeling.

Protein Dynamics to Define and Refine Disordered Protein Ensembles.

Intrinsically disordered proteins and unfolded proteins have fluctuating conformational ensembles that are fundamental to their biological function and impact protein folding, stability, and misfolding. Despite the importance of protein dynamics and conformational sampling, time-dependent data types are not fully exploited when defining and refining disordered protein ensembles. Here we introduce a computational framework using an elastic network model and normal-mode displacements to generate a dynamic disordered ensemble consistent with NMR-derived dynamics parameters, including transverse R2 relaxation rates and Lipari-Szabo order parameters (S2 values). We illustrate our approach using the unfolded state of the drkN SH3 domain to show that the dynamical ensembles give better agreement than a static ensemble for a wide range of experimental validation data including NMR chemical shifts, J-couplings, nuclear Overhauser effects, paramagnetic relaxation enhancements, residual dipolar couplings, hydrodynamic radii, single-molecule fluorescence Förster resonance energy transfer, and small-angle X-ray scattering.

Multifunctional nanoparticles as theranostic agents for therapy and imaging of breast cancer.

Over the last decade, there has been significant developments in nanotechnology, in particular for combined imaging and therapeutic applications (theranostics). The core or shell of nanoemulsions (NEs) can be loaded with various therapeutic agents, including drugs with low solubility for effective treatment, or various imaging agents for specific imaging modalities (e.g., MRI, fluorescence). In this work, perfluorohexane (PFH) NEs were synthesized for theranostic applications and were coupled to silica coated gold nanoparticles (scAuNPs) to increase the generation of PFH bubbles upon laser induced vaporization (i.e., optical droplet vaporization). The localized heat generated from the absorption properties of these nanoparticles (used to provide photoacoustic signals) can also be used to treat cancer without significantly damaging nearby healthy tissues. The theranostic potential of these PFH-NEs for contrast imaging of tumors and as a drug-delivery vehicle for therapeutic purposes were demonstrated for both in vitro and in vivo systems using a combination of photoacoustic, ultrasound and fluorescence imaging modalities. The ability of PFH-NEs to couple with scAuNPs, attach to the membranes of cancer cells and internalize within cancer cells, are encouraging for targeted chemotherapeutic applications for directly inducing cancer cell death via vaporization in clinical settings.

Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 µs) and slow (200-300 µs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1's ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

Extended Experimental Inferential Structure Determination Method in Determining the Structural Ensembles of Disordered Protein States.

Proteins with intrinsic or unfolded state disorder comprise a new frontier in structural biology, requiring the characterization of diverse and dynamic structural ensembles. We introduce a comprehensive Bayesian framework, the Extended Experimental Inferential Structure Determination (X-EISD) method, that calculates the maximum log-likelihood of a disordered protein ensemble. X-EISD accounts for the uncertainties of a range of experimental data and back-calculation models from structures, including NMR chemical shifts, J-couplings, Nuclear Overhauser Effects (NOEs), paramagnetic relaxation enhancements (PREs), residual dipolar couplings (RDCs), hydrodynamic radii (R h ), single molecule fluorescence Förster resonance energy transfer (smFRET) and small angle X-ray scattering (SAXS). We apply X-EISD to the joint optimization against experimental data for the unfolded drkN SH3 domain and find that combining a local data type, such as chemical shifts or J-couplings, paired with long-ranged restraints such as NOEs, PREs or smFRET, yields structural ensembles in good agreement with all other data types if combined with representative IDP conformers.

Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition.

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.

Bayesian counting of photobleaching steps with physical priors.

Counting fluorescence photobleaching steps is commonly used to infer the number n0 of monomeric units of individual oligomeric protein complexes or misfolded protein aggregates. We present a principled Bayesian approach for counting that incorporates the statistics of photobleaching. Our physics-based prior leads to a simple and efficient numerical scheme for maximum a posteriori probability (MAP) estimates of the initial fluorophore number n^0. Our focus here is on using a calibration to precisely estimate n^0, though our approach can also be used to calibrate the photophysics. Imaging noise increases with n^0, while bias is often introduced by temporal averaging. We examine the effects of fluorophore number n^0 of the oligomer or aggregate, lifetime photon yield µeff of an individual fluorophore, and exposure time Δt of each image frame in a time-lapse experiment. We find that, in comparison with standard ratiometric approaches or with a "change-point" step-counting algorithm, our MAP approach is both more precise and less biased.

Publisher Correction: Exocyst dynamics during vesicle tethering and fusion.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Biochemistry and Molecular Genetics and Breast Surgery, Ehime University Graduate School of Medicine, 7910295, Japan' and affiliation 3 incorrectly read 'Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Matsuyama 7910295, Japan.' These errors have now been corrected in both the PDF and HTML versions of the Article.

Exocyst dynamics during vesicle tethering and fusion.

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

Ligand-Induced Coupling between Oligomers of the M2 Receptor and the Gi1 Protein in Live Cells.

Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M2 muscarinic receptor and of Gi1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M2 and mCherry-M2) and G proteins (i.e., eGFP-Gαi1ß1γ2 and mCherry-Gαi1ß1γ2) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and Gi1 can exist as homo-oligomers. Oligomerization of differently tagged Gi1 decreased upon the activation of co-expressed wild-type M2 receptor by an agonist. Measurements on a tagged M2 receptor (M2-mCherry) and eGFP-Gαi1ß1γ2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M2(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gαi1ß1γ2, indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M2 receptor on the one hand and of Gi1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.

Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy.

In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.

Conformational Heterogeneity and FRET Data Interpretation for Dimensions of Unfolded Proteins.

A mathematico-physically valid formulation is required to infer properties of disordered protein conformations from single-molecule Förster resonance energy transfer (smFRET). Conformational dimensions inferred by conventional approaches that presume a homogeneous conformational ensemble can be unphysical. When all possible-heterogeneous as well as homogeneous-conformational distributions are taken into account without prejudgment, a single value of average transfer efficiency 〈E〉 between dyes at two chain ends is generally consistent with highly diverse, multiple values of the average radius of gyration 〈Rg〉. Here we utilize unbiased conformational statistics from a coarse-grained explicit-chain model to establish a general logical framework to quantify this fundamental ambiguity in smFRET inference. As an application, we address the long-standing controversy regarding the denaturant dependence of 〈Rg〉 of unfolded proteins, focusing on Protein L as an example. Conventional smFRET inference concluded that 〈Rg〉 of unfolded Protein L is highly sensitive to [GuHCl], but data from SAXS suggested a near-constant 〈Rg〉 irrespective of [GuHCl]. Strikingly, our analysis indicates that although the reported 〈E〉 values for Protein L at [GuHCl] = 1 and 7 M are very different at 0.75 and 0.45, respectively, the Bayesian Rg2 distributions consistent with these two 〈E〉 values overlap by as much as 75%. Our findings suggest, in general, that the smFRET-SAXS discrepancy regarding unfolded protein dimensions likely arise from highly heterogeneous conformational ensembles at low or zero denaturant, and that additional experimental probes are needed to ascertain the nature of this heterogeneity.