-
The FEBS Journal Oct 2017The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β... (Review)
Review
The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β parallel topology and hence it is highly relevant to illuminate how they fold. Here, we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These polypeptides tend to temporarily misfold during unassisted folding to their functionally active forms. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. As a result, flavodoxin-like proteins form intermediates that are off-pathway to native protein and several of these species are molten globules (MGs). Experiments suggest that the off-pathway species are of helical nature and that flavodoxin-like proteins have a nonconserved transition state that determines the rate of productive folding. Folding of flavodoxin from Azotobacter vinelandii has been investigated extensively, enabling a schematic construction of its folding energy landscape. It is the only flavodoxin-like protein of which cotranslational folding has been probed. New insights that emphasize differences between in vivo and in vitro folding energy landscapes are emerging: the ribosome modulates MG formation in nascent apoflavodoxin and forces this polypeptide toward the native state.
Topics: Azotobacter vinelandii; Escherichia coli; Escherichia coli Proteins; Flavodoxin; Gene Expression; Methyl-Accepting Chemotaxis Proteins; Models, Molecular; Protein Biosynthesis; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Isoforms; Thermodynamics
PubMed: 28380286
DOI: 10.1111/febs.14077 -
Neurobiology of Aging Dec 2018The assembly of Tau into abundant β-sheet-rich filaments characterizes human tauopathies. A pathological pathway leading from monomeric to filamentous Tau is believed...
The assembly of Tau into abundant β-sheet-rich filaments characterizes human tauopathies. A pathological pathway leading from monomeric to filamentous Tau is believed to be at the heart of these diseases. However, in Drosophila models of Tauopathy, neurodegeneration has been observed in the absence of abundant Tau filaments. Here we investigated the role of Tau assembly into β-sheets by expressing wild-type and Δ306-311 human Tau-383 in the retina and brain of Drosophila. We analyzed both lines for eye abnormalities, brain vacuolization, Tau phosphorylation and assembly, as well as climbing activity and survival. Flies expressing wild-type Tau-383 showed MC-1 staining, Tau hyperphosphorylation, and neurodegeneration. By contrast, flies expressing Δ306-311 Tau-383 had less MC-1 staining, reduced Tau hyperphosphorylation, and no detectable neurodegeneration. Their climbing ability and lifespan were similar to those of nontransgenic flies. Fluorescence spectroscopy after addition of Thioflavin T, a dye that interacts with β-sheets, showed no signal when Δ306-311 Tau-383 was incubated with heparin. These findings demonstrate that the assembly of Tau into β-sheets is necessary for neurodegeneration.
Topics: Animals; Animals, Genetically Modified; Behavior, Animal; Brain; Disease Models, Animal; Drosophila melanogaster; Humans; Neurodegenerative Diseases; Phosphorylation; Protein Conformation, beta-Strand; Retina; tau Proteins
PubMed: 30240946
DOI: 10.1016/j.neurobiolaging.2018.07.022 -
International Journal of Molecular... Aug 2022In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several...
In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases-such as atherosclerosis and amyloidosis-where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in β-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of β-sheet rich amyloid fibrils and α-helix-rich condensates.
Topics: Amyloid; Amyloidogenic Proteins; Apolipoprotein A-I; Circular Dichroism; Protein Conformation, beta-Strand; Protein Structure, Secondary
PubMed: 35955915
DOI: 10.3390/ijms23158780 -
Communications Biology Jun 2023γ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer's disease pathology and other transmembrane...
γ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer's disease pathology and other transmembrane proteins. Substrate-bound structures reveal a stable hybrid β-sheet immediately following the substrate scissile bond consisting of β1 and β2 from the enzyme and β3 from the substrate. Molecular dynamics simulations and enhanced sampling simulations demonstrate that the hybrid β-sheet stability is strongly correlated with the formation of a stable cleavage-compatible active geometry and it also controls water access to the active site. The hybrid β-sheet is only stable for substrates with 3 or more C-terminal residues beyond the scissile bond. The simulation model allowed us to predict the effect of Pro and Phe mutations that weaken the formation of the hybrid β-sheet which were confirmed by experimental testing. Our study provides a direct explanation why γ-secretase preferentially cleaves APP in steps of 3 residues and how the hybrid β-sheet facilitates γ-secretase proteolysis.
Topics: Amyloid Precursor Protein Secretases; Catalytic Domain; Protein Conformation, beta-Strand; Amyloid beta-Protein Precursor; Water Supply
PubMed: 37355752
DOI: 10.1038/s42003-023-05039-y -
Molecules (Basel, Switzerland) Feb 2021The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence... (Review)
Review
The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.
Topics: Amyloid; Amyloid beta-Peptides; Amyloidogenic Proteins; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Protein Aggregation, Pathological; Protein Conformation, beta-Strand; Single Molecule Imaging; Spectrometry, Fluorescence
PubMed: 33670093
DOI: 10.3390/molecules26040948 -
Nature Communications Jul 2022Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of >58,000...
Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ~100 fold-switching proteins. Though ostensibly rare, these proteins raise the question of how many uncharacterized proteins have shapeshifting-rather than fixed-secondary structures. Here, we use a comparative sequence-based approach to predict fold switching in the universally conserved NusG transcription factor family, one member of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and β-sheet folds. Our approach predicts that 24% of sequences in this family undergo similar α-helix ⇌ β-sheet transitions. While these predictions cannot be reproduced by other state-of-the-art computational methods, they are confirmed by circular dichroism and nuclear magnetic resonance spectroscopy for 10 out of 10 sequence-diverse variants. This work suggests that fold switching may be a pervasive mechanism of transcriptional regulation in all kingdoms of life.
Topics: Amino Acid Sequence; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Domains; Transcription Factors
PubMed: 35778397
DOI: 10.1038/s41467-022-31532-9 -
ACS Chemical Biology Jun 2018Since the proposal of Anfinsen's thermodynamic hypothesis in 1963, our understanding of protein folding and dynamics has gained significant appreciation of its nuance... (Review)
Review
Since the proposal of Anfinsen's thermodynamic hypothesis in 1963, our understanding of protein folding and dynamics has gained significant appreciation of its nuance and complexity. Intrinsically disordered proteins, chameleonic sequences, morpheeins, and metamorphic proteins have broadened the protein folding paradigm. Here, we discuss noncanonical protein folding patterns, with an emphasis on metamorphic proteins, and we review known metamorphic proteins that occur naturally and that have been engineered in the laboratory. Finally, we discuss research areas surrounding metamorphic proteins that are primed for future exploration, including evolution, drug discovery, and the quest for previously unrecognized metamorphs. As we enter an age where we are capable of complex bioinformatic searches and de novo protein design, we are primed to search for previously unrecognized metamorphic proteins and to design our own metamorphs to act as targeted, switchable drugs; biosensors; and more.
Topics: Animals; Bacteria; Humans; Intrinsically Disordered Proteins; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Folding; Protein Unfolding
PubMed: 29787234
DOI: 10.1021/acschembio.8b00276 -
Biomacromolecules Jun 2020Hydrogels' hydrated fibrillar nature makes them the material of choice for the design and engineering of 3D scaffolds for cell culture, tissue engineering, and...
Hydrogels' hydrated fibrillar nature makes them the material of choice for the design and engineering of 3D scaffolds for cell culture, tissue engineering, and drug-delivery applications. One particular class of hydrogels which has been the focus of significant research is self-assembling peptide hydrogels. In the present work, we were interested in exploring how fiber-fiber edge interactions affect the self-assembly and gelation properties of amphipathic peptides. For this purpose, we investigated two β-sheet-forming peptides, FEFKFEFK (F8) and KFEFKFEFKK (KF8K), the latter one having the fiber edges covered by lysine residues. Our results showed that the addition of the two lysine residues did not affect the ability of the peptides to form β-sheet-rich fibers, provided that the overall charge carried by the two peptides was kept constant. However, it did significantly reduce edge-driven hydrophobic fiber-fiber associative interactions, resulting in reduced tendency for KF8K fibers to associate/aggregate laterally and form large fiber bundles and consequently network cross-links. This effect resulted in the formation of hydrogels with lower moduli but faster dynamics. As a result, KF8K fibers could be aligned only under high shear and at high concentration while F8 hydrogel fibers were found to align readily at low shear and low concentration. In addition, F8 hydrogels were found to fragment at high concentration because of the high aggregation state stabilizing the fiber bundles, resulting in fiber breakage rather than disentanglement and alignment.
Topics: Hydrogels; Hydrophobic and Hydrophilic Interactions; Peptides; Protein Conformation, beta-Strand; Tissue Engineering
PubMed: 32275138
DOI: 10.1021/acs.biomac.0c00229 -
Bioconjugate Chemistry Aug 2021Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological...
Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom . Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).
Topics: Amino Acid Sequence; Biomimetic Materials; Diacylglycerol Kinase; Lipids; Models, Molecular; Nanostructures; Protein Conformation, beta-Strand; Silicon Dioxide
PubMed: 34288667
DOI: 10.1021/acs.bioconjchem.1c00260 -
Nature Chemical Biology May 2023Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel...
Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular β-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations.
Topics: Mice; Animals; Prions; Protein Conformation, beta-Strand; Amyloid
PubMed: 36646960
DOI: 10.1038/s41589-022-01229-7