Synthesis and characterization of high affinity fluorogenic α-synuclein probes.

Fluorescent small molecules are powerful tools for imaging α-synuclein pathology in vitro and in vivo. In this work, we explore benzofuranone as a potential scaffold for the design of fluorescent α-synuclein probes. These compounds have high affinity for α-synuclein, show fluorescent turn-on upon binding to fibrils, and display different binding to Lewy bodies, Lewy neurites and glial cytoplasmic inclusion pathologies in post-mortem brain tissue. These studies not only reveal the potential of benzofuranone compounds as α-synuclein specific fluorescent probes, but also have implications for the ways in which α-synucleinopathies are conformationally different and display distinct small molecule binding sites.

Cyclized NDGA modifies dynamic α-synuclein monomers preventing aggregation and toxicity.

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson's disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). To demonstrate its utility, we have applied CPX to study (1) trafficking of biologically relevant synthetic vesicles and (2) intracellular processes involved in transmission of α-synuclein (αS) pathology. Our results demonstrate that CPX can serve as a minimally perturbing probe for tracking the dynamics of biomolecules.

Using a FRET Library with Multiple Probe Pairs To Drive Monte Carlo Simulations of α-Synuclein.

We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson's disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of α-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of α-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.

Fluorescence spectroscopy reveals N-terminal order in fibrillar forms of α-synuclein.

The neuronal protein α-synuclein (αS) plays a key role in Parkinson's disease, forming inclusions termed Lewy bodies and Lewy neurites. Recent improvements in cryo-electron diffraction and solid state NMR (ssNMR) have led to the elucidation of the structures of peptides derived from the αS fibril core and full-length human αS in fibrils. Despite the valuable insight offered by these methods, there are still several questions about the structures' relevance to pathological aggregates. Herein, we present fluorescence data collected in vitro under the conditions which fibrils are typically assembled. Our data suggest that, in solution, fibrils are largely structured as observed by ssNMR. However, we observe significant disparities in the αS N-terminus as compared to ssNMR data, which provide insight on its important role in αS aggregation and fibril structure.

Multicolor protein FRET with tryptophan, selective coumarin-cysteine labeling, and genetic acridonylalanine encoding.

Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.

Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies.

Direct cell-to-cell transmission of proteopathic α-synuclein (α-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of α-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied α-syn seed species. Herein, we employed fluorescently labeled α-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized α-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous α-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous α-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of α-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.

Site-Specific Fluorescence Polarization for Studying the Disaggregation of α-Synuclein Fibrils by Small Molecules.

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinson's disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinson's disease.

Correction: Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis.

Correction for 'Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis' by Conor M. Haney, et al., Org. Biomol. Chem., 2016, 14, 1584-1592.

Thermodynamic origin of α-helix stabilization by side-chain cross-links in a small protein.

Peptide cross-linking has been widely explored as a means of constraining short sequences into stable folded conformations, most commonly α-helices. The prevailing hypothesis for the origin of helix stabilization is an entropic effect resulting from backbone pre-organization; however, obtaining direct evidence bearing on this hypothesis is challenging. Here, we compare the folding thermodynamics of a small helix-rich protein domain and analogues containing one of three common cross-linking motifs. Analysis of the folding free energy landscapes of linear vs. cyclized species reveal consistent trends in the effect of cyclization on folding energetics, as well as subtle differences based on the chemistry of the cross link. Stabilization in all three systems arises entirely from a reduction in the entropic penalty of folding that more than compensates for an enthalpic destabilization of the folded state.

Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis.

Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.

Multiply labeling proteins for studies of folding and stability.

Fluorescence spectroscopy is a powerful method for monitoring protein folding in real-time with high resolution and sensitivity, but requires the site-specific introduction of labels into the protein. The ability to genetically incorporate unnatural amino acids (Uaas) allows for the efficient synthesis of fluorescently labeled proteins with minimally perturbing fluorophores. Here, we describe recent uses of labeled proteins in dynamic structure determination experiments and advances in unnatural amino acid incorporation for dual site-specific fluorescent labeling. The advent of increasingly sophisticated bioorthogonal chemistry reactions and the diversity of Uaas available for incorporation will greatly enable protein folding and stability studies.

Receptor-templated stapling of intrinsically disordered peptide ligands.

We report here a chemoselective peptide "stapling" method that can be performed on ligand-receptor complexes in situ. An appropriately structured macrocyclic bis-oxime linkage is shown to improve the affinity of a peptide ligand for its native protein receptor. The presence of the receptor as a template to preorganize the ligand into its bioactive conformation is found to bias reaction outcomes, suggesting the potential application of the method for receptor-assisted selection of stapled peptides.

Dynamic covalent side-chain cross-links via intermolecular oxime or hydrazone formation from bifunctional peptides and simple organic linkers.

Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime-based and hydrazone-based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side-chain to side-chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle.

Oxime side-chain cross-links in an α-helical coiled-coil protein: structure, thermodynamics, and folding-templated synthesis of bicyclic species.

Covalent side-chain cross-links are a versatile method to control peptide folding, particularly when α-helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein-protein complex. A series of sequence variants of the dimeric coiled coil GCN4-p1 bearing oxime bridges at solvent-exposed positions were prepared and biophysically characterized. Triggered unmasking of a side-chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X-ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α-helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.

Promoting peptide α-helix formation with dynamic covalent oxime side-chain cross-links.

Covalent side-chain cross-linking has been shown to be a viable strategy to control peptide folding. We report here that an oxime side-chain linkage can elicit α-helical folds from peptides in aqueous solution. The bio-orthogonal bridge is formed rapidly under neutral buffered conditions, and the resulting cyclic oximes are capable of dynamic covalent exchange.

Identification of Hsp70 modulators through modeling of the substrate binding domain.

The design, synthesis and preliminary activity of small molecular weight modulators of the heat shock protein 70 (Hsp70) are described. The compounds provide a starting point for the synthesis of novel tools to decipher Hsp70 biology.