Alpha-synuclein structures probed by 5-fluorotryptophan fluorescence and 19F NMR spectroscopy

Alpha-synuclein, the main protein component of fibrillar deposits found in Parkinson's disease, is intrinsically disordered in vitro. Site-specific information on the protein conformation has been obtained by biosynthetic incorporation of an unnatural amino acid, 5-fluorotryptophan (5FW), into the recombinant protein. Using fluorescence and 19F NMR spectroscopy, we have characterized three proteins with 5FW at positions 4, 39, and 94. Steady-state emission spectra (maxima at 353 nm; quantum yields approximately 0.2) indicate that all three indole side chains are exposed to the aqueous medium. Virtually identical single-exponential excited-state decays (tau approximately 3.4 ns) were observed in all three cases. Single 19F NMR resonances were measured for W4, W39, and W94 at -49.0 +/- 0.1 ppm. Our analysis of the spectroscopic data suggests that the protein conformations are very similar in the regions near the three sites.     

Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

Transient lens spectroscopy of ultrafast internal conversion processes in citranaxanthin

The ultrafast internal conversion (IC) dynamics of the apocarotenoid citranaxanthin have been studied for the first time by means of two-color transient lens (TL) pump-probe spectroscopy. After excitation into the high-energy edge of the S2 band by a pump pulse at 400 nm, the subsequent intramolecular processes were probed at 800 nm. Experiments were performed in a variety of solvents at room temperature. Upper limits for the S2 lifetime tau2 on the order of 100-200 fs are estimated. The S1 lifetime tau1 varies only slightly between solvents (10-12 ps), and the only clear decrease is observed for methanol (8.5 ps). The findings are consistent with earlier results from transient absorption studies of other apocarotenoids and carotenoid ketones and transient lens experiments of C40 carbonyl carotenoids. Possible reasons for the observed weak solvent dependence of tau1 for citranaxanthin are discussed.     

Two‐Dimensional Crowding Uncovers a Hidden Conformation of α‐Synuclein

The intrinsically disordered protein (IDP), α‐synuclein (αS), is well‐known for phospholipid membrane binding‐coupled folding into tunable helical conformers. Here, using single‐molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction–folding landscape of αS can be tuned by two‐dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS–membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher‐affinity module remains membrane‐bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane‐binding IDPs with multiple binding modes.     

Cononsolvency-induced micellization kinetics of pyrene end-labeled diblock copolymer of N-isopropylacrylamide and oligo(ethylene glycol) methyl ether methacrylate studied by stopped-flow light-scattering and fluorescence

Cononsolvency-induced micellization kinetics of a pyrene end-labeled diblock copolymer of N-isopropylacrylamide and oligo(ethylene glycol) methyl ether methacrylate, Py-PNIPAM-b-POEGMA, was investigated in detail via a combination of stopped-flow light-scattering and fluorescence techniques. Upon a stopped-flow jump from pure methanol to proper methanol/water mixtures, scattered light intensity exhibited an initial increase and then stabilized out; whereas the time-dependence of monomer to excimer fluorescence intensity ratios (I E/I M) revealed an abrupt increase followed by a gradual decrease to plateau values. The dynamic traces of scattered intensity can be well fitted by double exponential functions, the obtained tau 1, scat and tau 2, scat can be ascribed to processes of forming quasi-equilibrium micelles and their relaxation into final equilibrium states, respectively. On the other hand, a triple exponential function was needed to fit the dynamic traces of I E/I M, leading to three characteristic relaxation times (tau 1, fluo, tau 2, fluo, and tau 3, fluo). It was found that the time scales of tau 1, scat and tau 2, scat obtained from stopped-flow light scattering were in general agreement with tau 2, fluo and tau 3, fluo obtained from stopped-flow fluorescence. Considering that excimer fluorescence is extremely sensitive to small aggregates, the newly detected fast process (tau 1, fluo) approximately 10 ms) by stopped-flow fluorescence should be ascribed to the early stage of micellization, i.e., the burst formation of small transient micelles, in which light scattering detection was still not sensitive enough. These small transient micelles fused and grew into quasi-equilibrium micelles, which then slowly relaxed into the final equilibrium state.     

A Phosphorylation‐Induced Turn Defines the Alzheimer’s Disease AT8 Antibody Epitope on the Tau Protein

Post mortem biochemical staging of Alzheimer’s disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer₂₀₂/pThr₂₀₅ region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr₂₀₅ residue to the amide proton of Gly₂₀₇, and is further stabilized by the two Arg residues opposing the pSer₂₀₂/pThr₂₀₅.     

Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif

The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif.     

Effect of oxygen on the formation and decay of stilbene radical cation during the resonant two-photon ionization

Formation and decay of radical cations of trans-stilbene and p-substituted trans-stilbenes (S.+) during the resonant two-photon ionization (TPI) of S in acetonitrile in the presence and absence of O(2) have been studied with laser flash photolysis using a XeCl excimer laser (308 nm, fwhm 25 ns). The transient absorption spectra of S.+ were observed with a peak around 470-490 nm. The formation quantum yield of S.+ (0.06-0.29) increased with decreasing oxidation potential (E(ox)) and increasing fluorescence lifetime (tau(f)) of S, except for trans-4-methoxystilbene which has the lowest E(ox) and longer tau(f) among S. The considerable low yield and fast decay in a few tens of nanoseconds time scale were observed for trans-4-methoxystilbene.+ in the presence of O(2), but not for other S.+ . It is suggested that formation of the ground-state complex between trans-4-methoxystilbene and O(2) and the distonic character of trans-4-methoxystilbene.+ with separation and localization of the positive charge on the oxygen of the p-methoxyl group and an unpaired electron on the beta-olefinic carbon are responsible for the fast reaction of trans-4-methoxystilbene.+ with O(2) or superoxide anion, leading to the considerable low yield and fast decay of trans-4-methoxystilbene.+ . The mechanism based on the transient absorption measurement of S.+ during the TPI is consistent with the relatively high oxidation efficiency of trans-4-methoxystilbene among S based on the product analysis during the photoinduced electron transfer in the presence of a photosensitizer such as 9,10-dicyanoanthracene and O(2) in acetonitrile.     

In Silico Analysis of Metabolites from Peruvian Native Plants as Potential Therapeutics against Alzheimer’s Disease

Background: Despite research on the molecular bases of Alzheimer’s disease (AD), effective therapies against its progression are still needed. Recent studies have shown direct links between AD progression and neurovascular dysfunction, highlighting it as a potential target for new therapeutics development. In this work, we screened and evaluated the inhibitory effect of natural compounds from native Peruvian plants against tau protein, amyloid beta, and angiotensin II type 1 receptor (AT1R) pathologic AD markers. Methods: We applied in silico analysis, such as virtual screening, molecular docking, molecular dynamics simulation (MD), and MM/GBSA estimation, to identify metabolites from Peruvian plants with inhibitory properties, and compared them to nicotinamide, telmisartan, and grapeseed extract drugs in clinical trials. Results: Our results demonstrated the increased bioactivity of three plants’ metabolites against tau protein, amyloid beta, and AT1R. The MD simulations indicated the stability of the AT1R:floribundic acid, amyloid beta:rutin, and tau:brassicasterol systems. A polypharmaceutical potential was observed for rutin due to its high affinity to AT1R, amyloid beta, and tau. The metabolite floribundic acid showed bioactivity against the AT1R and tau, and the metabolite brassicasterol showed bioactivity against the amyloid beta and tau. Conclusions: This study has identified molecules from native Peruvian plants that have the potential to bind three pathologic markers of AD.     

Does liquid–liquid phase separation drive peptide folding?

Proline–arginine (PR) dipeptide repeats have been shown to undergo liquid–liquid phase separation and are an example of a growing number of intrinsically disordered proteins that can assemble into membraneless organelles. These structures have been posited as nucleation sites for pathogenic protein aggregation. As such, a better understanding of the effects that the increased local concentration and volumetric crowding within droplets have on peptide secondary structure is necessary. Herein we use Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopy to show that formation of droplets by PR₂₀ accompanies changes in the amide-I spectra consistent with folding into poly-proline helical structures.

Two-dimensional infrared spectroscopy reveals folding of an intrinsically disordered peptide when sequestered into a model “membrane-less” organelle.     

Novel NMR Assignment Strategy Reveals Structural Heterogeneity in Solution of the nsP3 HVD Domain of Venezuelan Equine Encephalitis Virus

In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable α-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures.     

Disordered proteins and network disorder in network descriptions of protein structure, dynamics and function: hypotheses and a comprehensive review

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here we review the links between disordered proteins and the associated networks, and describe the consequences of local, mesoscopic and global network disorder on changes in protein structure and dynamics. We introduce a new classification of protein networks into 'cumulus-type', i.e., those similar to puffy (white) clouds, and 'stratus-type', i.e., those similar to flat, dense (dark) low-lying clouds, and relate these network types to protein disorder dynamics and to differences in energy transmission processes. In the first class, there is limited overlap between the modules, which implies higher rigidity of the individual units; there the conformational changes can be described by an 'energy transfer' mechanism. In the second class, the topology presents a compact structure with significant overlap between the modules; there the conformational changes can be described by 'multi-trajectories'; that is, multiple highly populated pathways. We further propose that disordered protein regions evolved to help other protein segments reach 'rarely visited' but functionally-related states. We also show the role of disorder in 'spatial games' of amino acids; highlight the effects of intrinsically disordered proteins (IDPs) on cellular networks and list some possible studies linking protein disorder and protein structure networks.     

Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53

The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.     

Ultrafast transient lens spectroscopy of various C40 carotenoids: lycopene, beta-carotene, (3R,3'R)-zeaxanthin, (3R,3'R,6'R)-lutein, echinenone, canthaxanthin, and astaxanthin

The ultrafast internal conversion (IC) dynamics of seven C(40) carotenoids have been investigated at room temperature in a variety of solvents using two-color transient lens (TL) pump-probe spectroscopy. We provide comprehensive data sets for the carbonyl carotenoids canthaxanthin, astaxanthin, and-for the first time-echinenone, as well as new data for lycopene, beta-carotene, (3R,3'R)-zeaxanthin and (3R,3'R,6'R)-lutein in solvents which have not yet been investigated in the literature. Measurements were carried out to determine, how the IC processes are influenced by the conjugation length of the carotenoids, additional substituents and the polarity of the solvent. TL signals were recorded at 800 nm following excitation into the high energy edge of the carotenoid S2 band at 400 nm. For the S2 lifetime solvent-independent upper limits on the order of 100-200 fs are estimated for all carotenoids studied. The S1 lifetimes are in the picosecond range and increase systematically with decreasing conjugation length. For instance, in the sequence canthaxanthin/echinenone/beta-carotene (13/12/11 double bonds) one finds tau1 approximately 5, 7.7 and 9 ps for the S1-->S0 IC process, respectively. Hydroxyl groups not attached to the conjugated system have no apparent influence on tau1, as observed for canthaxanthin/astaxanthin (tau1 approximately 5 ps in both cases). For all carotenoids studied, tau1 is found to be insensitive to the solvent polarity. This is particularly interesting in the case of echinenone, canthaxanthin and astaxanthin, because earlier measurements for other carbonyl carotenoids like, e.g., peridinin partly showed dramatic differences. The likely presence of an intramolecular charge transfer state in the excited state manifold of C40 carbonyl carotenoids, which is stabilized in polar solvents, has obviously no influence on the measured tau1.     

Replica exchange with guided annealing for accelerated sampling of disordered protein conformations

We critically examine a recently proposed convective replica exchange (cRE) method for enhanced sampling of protein conformation based on theoretical and numerical analysis. The results demonstrate that cRE and related replica exchange with guided annealing (RE‐GA) schemes lead to unbalanced exchange attempt probabilities and break detailed balance whenever the system undergoes slow conformational transitions (relative to the temperature diffusion timescale). Nonetheless, numerical simulations suggest that approximate canonical ensembles can be generated for systems with small conformational transition barriers. This suggests that RE‐GA maybe suitable for simulating intrinsically disordered proteins, an important class of newly recognized functional proteins. The efficacy of RE‐GA is demonstrated by calculating the conformational ensembles of intrinsically disordered kinase inducible domain protein. The results show that RE‐GA helps the protein to escape nonspecific compact states more efficiently and provides several fold speedups in generating converged and largely correct ensembles compared to the standard temperature RE. © 2014 Wiley Periodicals, Inc.     

Monitoring the Interaction of α-Synuclein with Calcium Ions through Exclusively Heteronuclear Nuclear Magnetic Resonance Experiments

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca²⁺ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.     

Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy

In-cell NMR provides a valuable means to assess how macromolecules, with concentrations up to 300 g/L in the cytoplasm, affect the structure and dynamics of proteins at atomic resolution. Here an intrinsically disordered protein, alpha-synuclein (alphaSN), and a globular protein, chymotrypsin inhibitor 2 (CI2) were examined by using in-cell NMR. High-resolution in-cell spectra of alphaSN can be obtained, but CI2 leaks from the cell and the remaining intracellular CI2 is not detectable. Even after stabilizing the cells from leakage by using alginate encapsulation, no CI2 signal is detected. From in vitro studies we conclude that this difference in detectability is the result of the differential dynamical response of disordered and ordered proteins to the changes of motion caused by the increased viscosity in cells.     

Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes

Proteins are constantly involved in the multitude of various interactions creating sophisticated networks which define and control all (or almost all) the biological processes taking place in any living organism. Intrinsically disordered proteins or regions play a number of crucial roles in mediating protein interactions. The lack of fixed structure protruding to the high level of intrinsic dynamics and almost unrestricted flexibility at various structure levels, being the major characteristics of intrinsically disordered proteins, provides them with unprecedented advantages over the ordered proteins. The binding modes attainable by disordered proteins are highly diverse, creating a multitude of unusual complexes. Although the majority of studied to date intrinsic disorder-based complexes are ordered or static entities originating due to the global or local disorder-to-order transitions, a new development is the discovery of dynamic complexes in which intrinsically disordered proteins continue to sample an ensemble of rapidly interconverting conformations mostly devoid of structure even in their bound state. The goal of this critical review is to illustrate binding plasticity of intrinsically disordered proteins by representing a portrait gallery of the disorder-based complexes (119 references).