1.
Efficient construction of disordered protein ensembles in a bayesian framework with optimal selection of conformations.
Source
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, Massachusetts 02139-4307, United States. ckfisher@fas.harvard.edu.
Abstract
Constructing an accurate model for the thermally accessible states of an Intrinsically Disordered Protein (IDP) is a fundamental problem in structural biology. This problem requires one to consider a large number of conformations in order to ensure that the model adequately represents the range of structures that the protein can adopt. Typically, one samples a wide range of structures in an attempt to obtain an ensemble that agrees with some pre-specified set of experimental data. However, models that contain more structures than the available experimental restraints are problematic as the large number of degrees of freedom in the ensemble leads to considerable uncertainty in the final model. We introduce a computationally efficient algorithm called Variational Bayesian Weighting with StructureSelection (VBWSS) for constructing a model for the ensemble of an IDP that contains a minimal number of conformations and, simultaneously, provides estimates for the uncertainty in properties calculated from the model. The algorithm is validated using reference ensembles and applied to construct an ensemble for the 140-residue IDP, monomeric α- synuclein.
[Selective stimulations and lesions of the rat brain nuclei as the models for research of the human sleep pathology mechanisms].
Abstract
Many complex behavioral phenomena such as sleep can not be explained without multidisciplinary experimental approach, and complementay approaches in the animal models "in vivo" and human studies. Electrophysiological, pharmacological, anatomical and immunohistochemical techniques, and particularly stereotaxically guided local nanovolume microinjection technique, enable us to selectively stimulate and lesion the brain nuclei or their specific neuronal subpopulation, and to reslove the mechanisms of certain brain structure regulatory role, and its afferent-efferent connectivity within the brain. Local stereotaxically guided nanovolume microinjection technique enable us to investigate in animals the brain nulcei functional topography with a resolution of < or = 10 microM, and at a level of 300 microM of effective radius within the brain tissue "in vivo". The advantage of local glutamate or DL- homocysteic acid microinjection stimulation or local excitotoxic (glutamate, ibotenic acid, IgG saporin) microinjection lesion over electrical stimulation/lesion of the same neuronal population are that they reduces the likelihood of activation/lesion of fibers of passage. Much of our knowledge of the sleep neuronal substrates is based on animal studies primarly in cat and rat. Selective pharmacological stimulation of the pedunculopontine tegmentum (PPT) in freely moving rat, using glutamate microinjection, proved that excitation of its cholinergic part is necessary for induction of wakefulness or REM (Datta S, 2001). Local nanovolume glutamate microinjection into PPT of anesthetized rats (Saponjić et al, 2003a) additionally evidenced P-wave and respiratory regulating neuronal subpopulation within the cholinergic compartment of PPT (apneogenic neuronal zone). Local microinjection of serotonin and noradrenaline into cholinergic PPT apneogenic zone evidenced their opposed impact through PPT on breathing, in contrast to their convergent regulatory role in behavioral state control (Saponjić et al., 2005a). Also, selective pharmacological stimulation by microinjection of DL-homocysteic acid defined four neuronal micro-circuitry approximately 500 microm in lenght of breathing-related neurons within the ventral respiratory group of medulla oblongata, which when stimulated produce different effects on respiratory rate, rhythm and amplitude, and on blood pressure. This study was the first high resolution study in order to understand anatomical and functional neuronal system organization (Monnier et al., 2003). Recently, local glutamate microinjection stimulation technique enabled detailed functional topography of respiratory, cardiovascular and pontine-wave responses within the PPT (Topchiy et al., 2010). Discovery of "flip-flop" switch for REM sleep control is based on the experiments in rats using local stereotaxically guided microinjection of excitotoxins (ibotenic acid, IgG saporin), and the anterograde and retrograde tracers for selective lesion, and identifying "REM-off" and "REM- on" regions and their afferent-efferent connections, and for identifying pathways for REM atonia and REM EEG activation (Lu et al., 2006). Recently, selective lesion of SLD part of "REM-on" region in rat established an animal model of RBD, as well as a selective ibotenic acid lesion of PC part of "REM-on" region abolished theta during REM (Lu et al., 200; Anaclet et al., 2010). Selective ablation targeted to pre-Bötzinger complex neurons of ventrolateral respiratory group of medulla in rat induced REM related respiratory disorder up to 10 days, when this respiratory disorder became spreaded to all sleep phases, and even during wakefulness, due to long-lasting intermitent hypoxia, and an increase of the threshold for hypoxia/hypercapnea induced arousal response (McKay et al., 2005). Human development, maturation, healthy aging and many neurological diseases are associated with profound changes in sleep/wake states distribution and with variety of the sleep-related behavioral disorders. Sleep and sleep-related respiratory disorders (insomnia, hypersomnia, parasomnias, excessive nocturnal motor activity, circadian sleep-wake rhythm disturbances, respiratory dysrhythmias, RBD) are very frequently unnoticed in patients with neurodegenerative diseases (Boeve et al., 2007; Whitwell et al., 2007). Alzheimer's and Parkinson's disease (AD, PD) are the most common neurodegenerative diseases, with prevalence of 0.5-1%; increasing to 1-3% for Parkinson, and up to 50% for Alzheimer's disease in ages over 69 (Nussbaum and Christopher, 2003). In spite of a long knowledge of their clinical description and brain pathology (lesions of the NB cholinergic neurons in basal forebrain, dopaminergic neurons in substantia nigra, etc.), they remain incurable with only limited success in temporal amelioration of their symptoms. Clinical symptoms first appear at 65-69 years on average, but there are indications that subclinical features may start many years earlier. Patients with REM-sleep behavior disorder (RBD) face close to a 20% 5-year risk of developing PD or dementia, and that risk rises to more than 40% after 10 years, and exceeds 50% after 12 years. Human studies evidenced that sleep/wake cycle disturbance, as no cognitive symptom of dementia, precedes on average 3 years before the clinical diagnosis of the AD (Simic et al., 2009), and that RBD, precedes as symptom the onset of motor and cognitive disturbances by years or decades. AD and PD involve the selective loss of specific neuronal populations within the brain. RBD in those patients reflects an underlying synucleinopathy, with presence of the alpha-synuclein protein pathology within the REM sleep-related regulatory structures of the dorsal midbrain and pons at the onset of disease, with ascending pattern of neurodegeneration progression from brainstem to basal areas of the brain (Whitwell et al., 2007; Simic et al., 2009: Raggi and Ferri, 2010). On the base of hypothesis that basal forebrain cholinergic system plays an important role in the etiology of the most common neurodegenerative diseases of elderly (AD, PD), the lesion of the nucleus basalis in rat presents the most utilized "in vivo" animal model to study the disorders of cortical cholinergic innervation, and its impact on higher central nervous system functions. Our knowledge of the neural substrates for sleep/wake states and sleep-related behavior disorders regulation in health and the diseases, over more than 50 years of sleep research, is based on animal models, pharmacotherapy, central nervous system lesions, and the neuropathological studies in humans. Today we have many complementary animal models of human sleep pathology, and further work in fundamental multidisciplinary and clinical research between sleep and neurodegenerative disease investigators is promising to enable us understand normal and abnormal sleep, and may provide new insights into preventive or disease-altering approaches for therapy. Obviously counseling and prevention of AD or PD would be highly enriched by the development of a practical, sensitive and reliable methodology of detecting those patients with RBD, or other sleep disorders, who are at risk for developing AD or PD.
- PMID:
- 22165729
- [PubMed - in process]
Effect of melatonin on α-synuclein self-assembly and cytotoxicity.
Source
Department of Neurology and Neurobiology and Aging, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.
Abstract
α-Synuclein (αS) assembly has been implicated as a critical step in the development of Lewy body diseases such as Parkinson's disease and dementia with Lewy bodies. Melatonin (Mel), a secretory product of the pineal gland, is known to have beneficial effects such as an antioxidant function and neuroprotection. To elucidate whether Mel has an antiassembly effect, here we used circular dichroism spectroscopy, photoinduced crosslinking of unmodified proteins, thioflavin S fluorescence, size exclusion chromatography, electron microscopy and atomic force microscopy to examine the effects of Mel on the αS assembly. We also examined the effects of Mel on αS-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism in αS-treated, primary neuronal cells. Initial studies revealed that Mel blocked αS fibril formation as well as destabilizing preformed αS fibrils. Subsequent evaluation of the assembly-stage specificity of the effect showed that Mel was able to inhibit protofibril formation, oligomerization, and secondary structure transitions. Importantly, Mel decreased αS-induced cytotoxicity. These data suggest a mechanism of action for Mel, inhibition of assembly of toxic polymers and protection of neurons from their effect.
Copyright © 2011 Elsevier Inc. All rights reserved.
STITCHER: Dynamic assembly of likely amyloid and prion β-structures from secondary structure predictions.
Source
Harvard/MIT Division of Health Science and Technology, Bioinformatics and Integrative Genomics, E25-519 Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142; MIT Computer Science and Artificial Intelligence Laboratory, The Stata Center, Cambridge, Massachusetts 02139.
Abstract
The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability-based prediction of discrete β-strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete β-strand pairs into complete amyloid β-structures. The STITCHER algorithm progressively 'stitches' strand-pairs into full β-sheets based on a novel free-energy model, incorporating experimentally observed amino-acid side-chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel β-sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer's amyloid beta peptide and the Podospora anserina Het-s prion. Predictions of the HET-s homolog HET-S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N-terminal structural stability enabled by tyrosine ladders, and C-terminal heterogeneity. Predictions for the Rnq1 prion and alpha-synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. Proteins 2011. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Membrane proteins in four acts: Function precedes structure determination.
Source
Department of Biological Sciences, Purdue University, Hall of Structural Biology, 240 Hockmeyer Hall, West Lafayette, IN 47907-1354, USA.
Abstract
Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.
Copyright © 2011. Published by Elsevier Inc.
Explaining the Structural Plasticity of α-Synuclein.
Source
Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139-4307, United States.
Abstract
Given that α-synuclein has been implicated in the pathogenesis of several neurodegenerative disorders, deciphering thestructure of this protein is of particular importance. While monomeric α-synuclein is disordered in solution, it can form aggregates rich in cross-β structure, relatively long helical segments when bound to micelles or lipid vesicles, and a relatively ordered helical tetramer within the native cell environment. To understand the physical basis underlying this structural plasticity, we generated an ensemble for monomeric α-synuclein using a Bayesian formalism that combines data from NMR chemical shifts, RDCs, and SAXS with molecular simulations. An analysis of the resulting ensemble suggests that a non-negligible fraction of the ensemble (0.08, 95% confidence interval 0.03-0.12) places the minimal toxic aggregation-prone segment in α-synuclein, NAC(8-18), in a solvent exposed and extended conformation that can form cross-β structure. Our data also suggest that a sizable fraction of structures in the ensemble (0.14, 95% confidence interval 0.04-0.23) contains long-range contacts between the N- and C-termini. Moreover, a significant fraction of structures that contain these long-range contacts also place the NAC(8-18) segment in a solvent exposed orientation, a finding in contrast to the theory that such long-range contacts help to prevent aggregation. Lastly, our data suggest that α-synuclein samples structures with amphipathic helices that can self-associate via hydrophobic contacts to form tetrameric structures. Overall, these observations represent a comprehensive view of the unfolded ensemble of monomeric α-synuclein and explain how different conformations can arise from the monomeric protein.
A soluble α-synuclein construct forms a dynamic tetramer.
Source
Department of Biochemistry and Molecular Biology, and Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Abstract
A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.
Purification of hsp104, a protein disaggregase.
Source
Department of Biochemistry and Biophysics, University of Pennsylvania, USA.
Abstract
Hsp104 is a hexameric AAA+ protein(1) from yeast, which couples ATP hydrolysis to protein disaggregation (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2). This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils. Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease as well as amyloid forms of PrP. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease and Huntington's disease. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors. To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102 kDa protein with a pI of -5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His(6)-tag removal compared to a previous purification method from E. coli. Moreover, our protocol is more facile and convenient than two more recent protocols.
Multiple system atrophy: a clinical and neuropathological perspective.
Source
Department of Neurosciences, University of California, San Diego/La Jolla, CA, USA, USA.
Abstract
Multiple system atrophy (MSA) is a neurodegenerative disease involving motor abnormalities that include akinesia, rigidity and postural instability. While improved diagnostic criteria have aided the accurate diagnosis of MSA, our understanding of the neuropathological aspects underlying MSA was bolstered by the identification of α-synuclein (α-syn) as the primary constituent of the abnormal protein aggregates observed in the brains of MSA patients. The generation of transgenic animal models of MSA coupled with an increasing understanding of the biochemical structureand function of α-syn has highlighted a number of key pathological pathways thought to underlie the neurodegeneration observed in MSA. This review summarizes key findings in the field, discusses current areas of debate, and describes current experimental approaches towards disease-modifying therapies.
Copyright © 2011 Elsevier Ltd. All rights reserved.
The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones.
Source
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-7520, USA.
Abstract
The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:β-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α-synuclein-mediated neurotoxicity and fibrillation is considered.
Copyright © 2011 The Protein Society.
Membrane Curvature Sensing by Amphipathic Helices: A SINGLE LIPOSOME STUDY USING α-SYNUCLEIN AND ANNEXIN B12.
Source
From the Bionanotechnology and Nanomedicine Laboratory, Department of Neuroscience and Pharmacology.
Abstract
Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.
The Effects of Regularly Spaced Glutamine Substitutions on Alpha-Helical Peptide Structures. A DFT/ONIOM Study.
Source
Department of Chemistry, City University of New York - Hunter College and the Graduate School, 695 Park Avenue, New York NY 10065.
Abstract
The side-chains of the residues of glutamine (Q) and asparagine (N) contain amide groups. These can H-bond to each other in patterns similar to those of the backbone amides in α-helices. We show that mutating multiple Q's for alanines (A's) in a polyalanine helix stabilizes the helical structure, while similar mutations with multiple N's do not. We suggest that modification of peptides by incorporating Q's in such positions can make more robust helices that can be used to test the effects of secondary structures in biochemical experiments linked to proteins with variable structures such as tau and α-synuclein.
- PMID:
- 21927063
- [PubMed]
- PMCID: PMC3171806
- [Available on 2012/8/25]
α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation.
Source
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract
Parkinson's disease is the second most common neurodegenerative disorder. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson's disease. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites, the cytopathological hallmarks of Parkinson's disease and related disorders called synucleinopathies. α-Synuclein has long been defined as a 'natively unfolded' monomer of about 14 kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58 kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson's disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.
Comment in
- PMID:
- 21841800
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3166366
- [Available on 2012/3/1]
EPR in Protein Science : Intrinsically Disordered Proteins.
Source
Department of Chemistry, Zukunftskolleg, and Konstanz Research School Chemical Biology, University of Konstanz, 78457, Konstanz, Germany, malte.drescher@uni-konstanz.de.
Abstract
Intrinsically disordered proteins (IDPs) form a unique protein category characterized by the absence of a well-definedstructure and by remarkable conformational flexibility. Electron Paramagnetic Resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) is amongst the most suitable methods to unravel their structure and dynamics. This review summarizes the tremendous methodological developments in the area of SDSL EPR and its applications in protein research. Recent results on the intrinsically disordered Parkinson's disease protein α-synucleinillustrate that the method has gained increasing attention in IDP research. SDSL EPR has now reached a level where broad application in this rapidly advancing field is feasible.
Biophysics of α-synuclein membrane interactions.
Abstract
Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.
Copyright © 2011. Published by Elsevier B.V.
Computer modeling of nitroxide spin labels on proteins.
Source
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089; Department of Biochemistry, University of Southern California, Los Angeles, CA 90033-9151.
Abstract
Electron paramagnetic resonance using site-directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r(2) = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35-44, 2012.
Copyright © 2011 Wiley Periodicals, Inc.
Structural role of compensatory amino acid replacements in the α-synucleinprotein.
Source
International School for Advanced Studies, 34136 Trieste, Italy.
Abstract
A subset of familial Parkinson's disease (PD) cases is associated with the presence of disease-causing point mutations in human α-synuclein [huAS(wt)], including A53T. Surprisingly, the human neurotoxic amino acid 53T is present in non-primate, wild-type sequences of α-synucleins, including that expressed by mice [mAS(wt)]. Because huAS(A53T) causes neurodegeneration when expressed in rodents, the amino acid changes between the wild-type human protein [huAS(wt)] and mAS(wt) might act as intramolecular suppressors of A53T toxicity in the mouse protein, restoring its physiological structure and function. The lack of structural information for mAS(wt) in aqueous solution has prompted us to conduct a comparative molecular dynamics study of huAS(wt), huAS(A53T), and mAS(wt) in water at 300 K. The calculations are based on an ensemble of nuclear magnetic resonance-derived huAS(wt) structures. huAS(A53T) turns out to be more flexible and less compact than huAS(wt). Its central (NAC) region, involved in fibril formation by the protein, is more solvent-exposed than that of the wild-type protein, in agreement with nuclear magnetic resonance data. The compactness of mAS(wt) is similar to that of the human protein. In addition, its NAC region is less solvent-exposed and more rigid than that of huAS(A53T). All of these features may be caused by an increase in the level of intramolecular interactions on passing from huAS(A53T) to mAS(wt). We conclude that the presence of "compensatory replacements" in the mouse protein causes a significant change in the protein relative to huAS(A53T), restoring features not too dissimilar to those of the human protein.
Electron paramagnetic resonance spectroscopy measures the distance between the external β-strands of folded α-synuclein in amyloid fibrils.
Source
RG Electron Spin Resonance Spectroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Abstract
The misfolding of α-synuclein (αS) to a cross-β-sheet amyloid structure is associated with pathological conditions in Parkinson's and other neurodegenerative diseases. Using pulse electron paramagnetic resonance spectroscopy combined with a cross-labeling strategy involving four double mutants, we were able to determine the intramolecular distance between the extremal β-strands. The distance of 4.5 ± 0.5 nm is in good agreement with the dimensions of a protofilament reported by other low-resolution techniques, such as x-ray scattering and atomic force microscopy.
Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.
- PMID:
- 21723808
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3127190
- [Available on 2012/7/6]
The A53T mutation is key in defining the differences in the aggregation kinetics of human and mouse α-synuclein.
Source
Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, New Jersey 08854, United States.
Abstract
Despite a 95% sequence similarity, the aggregation of human and mouse α-synuclein is remarkably different, as the human form is slower than the mouse form in forming fibrils but is associated with Parkinson's disease in both humans and transgenic mice. Here, the amino acid code underlying these differences is investigated by comparing the lag times, growth rates, and secondary structure propensities of a systematic series of eight human-mouse chimeras. Fluorescence analysis of these variants shows that the A53T substitution dominates the growth kinetics, while the lag phase is affected by a combination of the A53T and S87N substitutions. The secondary structure propensities derived from an NMR chemical shift analysis of the monomeric forms of the human-mouse variants enable us to establish a link between the changes in the conformational properties in the region of position 53 upon mutation and the corresponding changes in growth rates. These results suggest that the presence of an alanine residue at position 53 may be an evolutionary adaptation to minimize Parkinson's disease in humans and indicates that effective drug development efforts may be directed to target this N-terminal region of the sequence.
- PMID:
- 21721555
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3205953
- [Available on 2012/8/31]
Structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites.
Source
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Abstract
α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD-A30P, E46K and A53T-are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.
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