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1.
Int J Mol Sci ; 22(19)2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34639093

RESUMO

Aggregation of ß2 microglobulin (ß2m) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type protein and its truncated variant, ΔN6 ß2m, in haemo-dialysed patients. A second form of familial systemic amyloidosis caused by the ß2m variant, D76N, results in amyloid deposits in the viscera, without renal dysfunction. Although the folding and misfolding mechanisms of ß2 microglobulin have been widely studied in vitro and in vivo, we lack a comparable understanding of the molecular mechanisms underlying toxicity in a cellular and organismal environment. Here, we established transgenic C. elegans lines expressing wild-type (WT) human ß2m, or the two highly amyloidogenic naturally occurring variants, D76N ß2m and ΔN6 ß2m, in the C. elegans bodywall muscle. Nematodes expressing the D76N ß2m and ΔN6 ß2m variants exhibit increased age-dependent and cell nonautonomous proteotoxicity associated with reduced motility, delayed development and shortened lifespan. Both ß2m variants cause widespread endogenous protein aggregation contributing to the increased toxicity in aged animals. We show that expression of ß2m reduces the capacity of C. elegans to cope with heat and endoplasmic reticulum (ER) stress, correlating with a deficiency to upregulate BiP/hsp-4 transcripts in response to ER stress in young adult animals. Interestingly, protein secretion in all ß2m variants is reduced, despite the presence of the natural signal sequence, suggesting a possible link between organismal ß2m toxicity and a disrupted ER secretory metabolism.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Estresse do Retículo Endoplasmático , Longevidade , Mutação , Agregados Proteicos , Resposta a Proteínas não Dobradas , Microglobulina beta-2/toxicidade , Animais , Caenorhabditis elegans/genética , Resposta ao Choque Térmico , Humanos , Microglobulina beta-2/genética
3.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
4.
Nat Chem ; 13(5): 397-399, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33931750
5.
AIChE J ; 67(3): e17101, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33776061

RESUMO

Determining the structure of the (oligomeric) intermediates that form during the self-assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo-induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid-forming peptide, Aß16-22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group-Fmoc(TFMD)Phe-was incorporated into the sequence (Aß*16-22). Electrospray ionization ion-mobility spectrometry mass-spectrometry (ESI-IMS-MS) analysis of the PIC products confirmed that Aß*16-22 forms assemblies with the monomers arranged as anti-parallel, in-register ß-strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aß16-22 assembles through a single-step nucleation into a ß-sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aß16-22 self-assembly processes, paving the way to explore the self-assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.

6.
J Am Soc Mass Spectrom ; 32(7): 1583-1592, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-33586970

RESUMO

NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein ß2-microglobulin (ß2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting methods, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), in conjunction with ion mobility-mass spectrometry, to probe the differences in conformational dynamics of the two proteins. Using HDX-MS, a clear difference in HDX protection is observed between these two proteins in the E-F loop (residues 70-77) which contains the D76N substitution, with a significantly higher deuterium uptake being observed in the variant protein. Conversely, following FPOP-MS only minimal differences in the level of oxidation between the two proteins are observed in the E-F loop region, suggesting only modest side-chain movements in that area. Together the HDX-MS and FPOP-MS data suggest that a tangible perturbation to the hydrogen-bonding network in the E-F loop has taken place in the D76N variant and furthermore illustrate the benefit of using multiple complementary footprinting methods to address subtle, but possibly biologically important, differences between highly similar proteins.

7.
Science ; 371(6531)2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33602829

RESUMO

Transmembrane ß-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow ß-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.


Assuntos
Simulação por Computador , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica em Folha beta , Engenharia de Proteínas , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Micelas , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica
8.
J Mol Biol ; 433(8): 166878, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33610557

RESUMO

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aß) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson's disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-ß sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's disease.


Assuntos
Benzopiranos/química , Benzopiranos/farmacologia , Encéfalo/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Conformação Molecular , Simulação de Acoplamento Molecular , Neurônios , alfa-Sinucleína/toxicidade
9.
Chem Rev ; 121(3): 1845-1893, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33427465

RESUMO

The possible link between hIAPP accumulation and ß-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, ß-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Proteostase , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Fatores de Risco
10.
Biophys Chem ; 268: 106505, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33220582

RESUMO

Oligomers which form during amyloid fibril assembly are considered to be key contributors towards amyloid disease. However, understanding how such intermediates form, their structure, and mechanisms of toxicity presents significant challenges due to their transient and heterogeneous nature. Here, we discuss two different strategies for addressing these challenges: use of (1) methods capable of detecting lowly-populated species within complex mixtures, such as NMR, single particle methods (including fluorescence and force spectroscopy), and mass spectrometry; and (2) chemical and biological tools to bias the amyloid energy landscape towards specific oligomeric states. While the former methods are well suited to following the kinetics of amyloid assembly and obtaining low-resolution structural information, the latter are capable of producing oligomer samples for high-resolution structural studies and inferring structure-toxicity relationships. Together, these different approaches should enable a clearer picture to be gained of the nature and role of oligomeric intermediates in amyloid formation and disease.


Assuntos
Amiloide/metabolismo , Amiloide/análise , Amiloidose/metabolismo , Animais , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Agregados Proteicos , Multimerização Proteica
11.
Front Mol Neurosci ; 13: 609073, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33324164

RESUMO

Amyloid plaques are a pathological hallmark of Alzheimer's disease. The major component of these plaques are highly ordered amyloid fibrils formed by amyloid-ß (Aß) peptides. However, whilst Aß amyloid fibril assembly has been subjected to detailed and extensive analysis in vitro, these studies may not reproduce how Aß fibrils assemble in the brain. This is because the brain represents a highly complex and dynamic environment, and in Alzheimer's disease multiple cofactors may affect the assembly of Aß fibrils. Moreover, in vivo amyloid plaque formation will reflect the balance between the assembly of Aß fibrils and their degradation. This review explores the roles of microglia as cofactors in Aß aggregation and in the clearance of amyloid deposits. In addition, we discuss how infection may be an additional cofactor in Aß fibril assembly by virtue of the antimicrobial properties of Aß peptides. Crucially, by understanding the roles of microglia and infection in Aß amyloid fibril assembly it may be possible to identify new therapeutic targets for Alzheimer's disease.

12.
Front Neurosci ; 14: 611285, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33335475

RESUMO

Amyloid proteins are involved in many neurodegenerative disorders such as Alzheimer's disease [Tau, Amyloid ß (Aß)], Parkinson's disease [alpha-synuclein (αSyn)], and amyotrophic lateral sclerosis (TDP-43). Driven by the early observation of the presence of ordered structure within amyloid fibrils and the potential to develop inhibitors of their formation, a major goal of the amyloid field has been to elucidate the structure of the amyloid fold at atomic resolution. This has now been achieved for a wide variety of sequences using solid-state NMR, microcrystallography, X-ray fiber diffraction and cryo-electron microscopy. These studies, together with in silico methods able to predict aggregation-prone regions (APRs) in protein sequences, have provided a wealth of information about the ordered fibril cores that comprise the amyloid fold. Structural and kinetic analyses have also shown that amyloidogenic proteins often contain less well-ordered sequences outside of the amyloid core (termed here as flanking regions) that modulate function, toxicity and/or aggregation rates. These flanking regions, which often form a dynamically disordered "fuzzy coat" around the fibril core, have been shown to play key parts in the physiological roles of functional amyloids, including the binding of RNA and in phase separation. They are also the mediators of chaperone binding and membrane binding/disruption in toxic amyloid assemblies. Here, we review the role of flanking regions in different proteins spanning both functional amyloid and amyloid in disease, in the context of their role in aggregation, toxicity and cellular (dys)function. Understanding the properties of these regions could provide new opportunities to target disease-related aggregation without disturbing critical biological functions.

13.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
14.
J Am Chem Soc ; 142(49): 20845-20854, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33253560

RESUMO

Protein-protein interactions (PPIs) are involved in many of life's essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein ß2-microglobulin (ß2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped, off-pathway oligomers using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of "post-translational chemical modification" as a tool to study biological molecular mechanisms.


Assuntos
Proteínas Amiloidogênicas/química , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Cristalografia por Raios X , Dissulfetos/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo
15.
Nat Struct Mol Biol ; 27(11): 1094, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33037421

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

16.
Nat Struct Mol Biol ; 27(11): 1048-1056, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32929282

RESUMO

Aggregation of the peptide hormone amylin into amyloid deposits is a pathological hallmark of type-2 diabetes (T2D). While no causal link between T2D and amyloid has been established, the S20G mutation in amylin is associated with early-onset T2D. Here we report cryo-EM structures of amyloid fibrils of wild-type human amylin and its S20G variant. The wild-type fibril structure, solved to 3.6-Å resolution, contains two protofilaments, each built from S-shaped subunits. S20G fibrils, by contrast, contain two major polymorphs. Their structures, solved at 3.9-Å and 4.0-Å resolution, respectively, share a common two-protofilament core that is distinct from the wild-type structure. Remarkably, one polymorph contains a third subunit with another, distinct, cross-ß conformation. The presence of two different backbone conformations within the same fibril may explain the increased aggregation propensity of S20G, and illustrates a potential structural basis for surface-templated fibril assembly.


Assuntos
Amiloide/genética , Diabetes Mellitus Tipo 2/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Amiloide/química , Amiloide/ultraestrutura , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Modelos Moleculares , Mutação Puntual , Conformação Proteica
17.
Lancet Neurol ; 19(10): 872-878, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32949547

RESUMO

Studies in experimental animals show transmissibility of amyloidogenic proteins associated with prion diseases, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Although these data raise potential concerns for public health, convincing evidence for human iatrogenic transmission only exists for prions and amyloid ß after systemic injections of contaminated growth hormone extracts or dura mater grafts derived from cadavers. Even though these procedures are now obsolete, some reports raise the possibility of iatrogenic transmission of amyloid ß through putatively contaminated neurosurgical equipment. Iatrogenic transmission of amyloid ß might lead to amyloid deposition in the brain parenchyma and blood vessel walls, potentially resulting in cerebral amyloid angiopathy after several decades. Cerebral amyloid angiopathy can cause life-threatening brain haemorrhages; yet, there is no proof that the transmission of amyloid ß can also lead to Alzheimer's dementia. Large, long-term epidemiological studies and sensitive, cost-efficient tools to detect amyloid are needed to better understand any potential routes of amyloid ß transmission and to clarify whether other similar proteopathic seeds, such as tau or α-synuclein, can also be transferred iatrogenically.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Doenças Neurodegenerativas/metabolismo , Vigilância da População , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fatores de Risco
18.
J Biol Chem ; 295(35): 12474-12484, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661194

RESUMO

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.


Assuntos
Amiloide/química , Simulação de Dinâmica Molecular , Agregados Proteicos , Microglobulina beta-2/química , Substituição de Aminoácidos , Amiloide/genética , Cristalografia por Raios X , Humanos , Mutação de Sentido Incorreto , Microglobulina beta-2/genética
19.
Protein Sci ; 29(8): 1851-1857, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32557917

RESUMO

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yield inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein, and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualization of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.


Assuntos
Modelos Moleculares , Proteínas/química , Software , Conformação Proteica
20.
Nano Lett ; 20(7): 5553-5561, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32559088

RESUMO

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (ß-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein ß-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.


Assuntos
Nanoporos , Amiloide , DNA , alfa-Sinucleína/genética
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