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.

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.

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.

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.

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.