Partial Folding Research Articles

Long-range Regulation of Partially Folded Amyloidogenic Peptides

Neurodegeneration involves abnormal aggregation of intrinsically disordered amyloidogenic peptides (IDPs), usually mediated by hydrophobic protein-protein interactions. There is mounting evidence that formation of α-helical intermediates is an early event during self-assembly of amyloid-β42 (Aβ42) and α-synuclein (αS) IDPs in Alzheimer’s and Parkinson’s disease pathogenesis, respectively. However, the driving force behind on-pathway molecular assembly of partially folded helical monomers into helical oligomers assembly remains unknown. Here, we employ extensive molecular dynamics simulations to sample the helical conformational sub-spaces of monomeric peptides of both Aβ42 and αS. Our computed free energies, population shifts, and dynamic cross-correlation network analyses reveal a common feature of long-range intra-peptide modulation of partial helical folds of the amyloidogenic central hydrophobic domains via concerted coupling with their charged terminal tails (N-terminus of Aβ42 and C-terminus of αS). The absence of such inter-domain fluctuations in both fully helical and completely unfolded (disordered) states suggests that long-range coupling regulates the dynamicity of partially folded helices, in both Aβ42 and αS peptides. The inter-domain coupling suggests a form of intra-molecular allosteric regulation of the aggregation trigger in partially folded helical monomers. This approach could be applied to study the broad range of amyloidogenic peptides, which could provide a new path to curbing pathogenic aggregation of partially folded conformers into oligomers, by inhibition of sites far from the hydrophobic core.

Architecture and evolution of an extensionally-inverted thrust (Mt. Tancia Thrust, Central Apennines): Geological, structural, geochemical, and K–Ar geochronological constraints

Fault inversion may lead to significant obliteration of earlier tectonic structures, thus preventing the straightforward interpretation of the complete kinematics and deformation history of faults.We adopt a multidisciplinary approach to: (1) reconstruct the tectonic evolution through space and in time of the extensionally-inverted Mt. Tancia Thrust (Central Apennines, Italy); (2) understand the deformation mechanisms and the transition and reactivation of compressional structures during negative tectonic inversion; and (3) constrain the origin of fluids involved during tectonic processes.To this end, we combined: (1) detailed geological mapping and multiscale structural analysis; (2) illite-smectite paleothermal indicators; (3) C, O, and clumped isotopes on calcite mineralizations and (4) K–Ar dating of authigenic and/or syn-kinematic illite from the Mt. Tancia Thrust fault rocks. We show that shortening occurred between ~9 and ~7 Ma, during possibly multiple events of fluid overpressure and shear rupturing involving fluids entrapped over long term within the host rocks. Post-compressive tectonic inversion occurred at ~3 Ma under fluid pressure fluctuations during shear events with an input of meteoric-derived fluids. Tectonic inversion is spatially confined within the first few metres below the thrust surface in a volume dominated by the partial overprinting, folding, transposition, and re-utilization of the earlier compressional fabric.

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment

The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal–epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein’s triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain’s folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly.

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Partial Vocal Fold Paresis

Partial Vocal Fold Paresis

Cationic Copolymers Act As Chaperones of a Membrane-Active Peptide: Influence on Membrane Selectivity.

Membrane-active peptides have potential as drug delivery tools for control of lipid bilayer structures in cells and liposomes. In a previous study, we reported that a cationic comb-type copolymer, poly(allylamine)-graft-dextran (PAA-g-Dex), forms a soluble interpolyelectrolyte complex with an anionic peptide, E5, and enhances its membrane-disrupting activity. Furthermore, the E5/PAA-g-Dex complex augments the cellular membrane permeability of other proteins. In this study, the affinities of the E5/PAA-g-Dex complex for lipid membranes with various compositions were determined. Secondary structure analysis of E5 and analyses of binding of E5 to liposomes revealed that lipid composition strongly influenced the interaction. No significant folding of E5 alone was observed at either pH 5.4 or pH 7.4 and folding into the functional conformation, which is both N-terminal and C-terminal helix, was observed only at pH 5.4 in the presence of liposomes having liquid-disordered phase (Ld). PAA-g-Dex induced partial folding of E5, presumably at C-terminus, at both pH 5.4 and pH 7.4. Folding of E5 into the functional structure was induced by the addition of liposomes having Ld phases at either pH 5.4 or pH 7.4. A leakage assay showed that PAA-g-Dex enhanced the membrane-permeabilizing activity of E5 by promoting the adsorption of E5 onto the surface of liposomes and/or E5 association with the lipid bilayer. These results indicated that E5 activated by PAA-g-Dex destabilizes the lipid membrane having Ld phase even when the lipid membrane has a heterogeneous phase separated structure. Hence, PAA-g-Dex serves as a chaperone for E5 without altering its membrane selectivity. The chaperoning activity of this comb-type copolymer may activate other ionic peptides with unstable structures and low solubility.

Multimodal imaging in posterior microphthalmos

PurposeTo evaluate the multimodal imaging including optical coherence tomography angiography (OCTA) findings in patients with posterior microphthalmos (PM). MethodsIn an observational case series, four eyes of two patients, eight and twenty-three years old, with clinical proven PM underwent complete ophthalmic examination, including refraction, fluorescein angiography, optical coherence tomography (OCT), OCTA, B-scan ultrasonography, axial length measurement using IOL Master optical measuring, and Pentacam evaluation. ResultsBoth patients were high hyperopic with partial thickness retinal fold in macula, retinoschisis, and foveal hypoplasia. Axial length was less than 17 mm with scleral thickening in all eyes. OCTA showed absence of foveal avascular zone (FAZ) in both superficial and deep capillary plexuses. Pentacam showed corneal steepness, shallow anterior chamber, and low anterior chamber volume. ConclusionOCTA findings showed absence of avascular zone in both superficial and deep capillary plexuses, while OCT shows partial thickness retinal fold and retinoschisis.

Single Molecule Snapshots of Riboswitch Conformational Change and RNA Switch Based Biosensing on a Nanopore Maglet Device

Riboswitches are a class of biosensory-regulatory segments of messenger-RNAs. Their binding with specific cellular ligands e.g. metabolic intermediates, can induce a conformational change in riboswitch's tertiary structure, ultimately programming the production of the proteins encoded by the mRNA. Precision tools to explore ligand-programmed riboswitch folding/unfolding mechanism are important in both developing riboswitch-targeted antibiotic intervention and RNA switch-based biosensors. Our nanopore weak-interaction spectroscopy can snapshot a single RNA riboswitch's folding/unfolding pathway at single base-pair resolution. Different from thermodynamic approaches and force microscopies (e.g. AFM and optic tweeze), the unique feature of nanopore is unfolding along the backbone of RNA-Vectorial Unfolding. This tool enables us to discriminate RNA unfolding pathway in both 3’>5’ and 5’>3’ directions, dissect the non-canonical triple-strand conformations and interactions that control the RNA stability, and many interconnected folding intermediates. Based on these findings, we further develop a nanopore maglet automatic machine that can “lock” a single riboswitch (preQ1) molecule in the nanopore. The locked single RNA can be electrically driven to repeatedly move back and forth to transform among different conformations: partial folding→ligand binding →fully folding→Unfolding. We can probe the folding change of riboswitch upon ligand (preQ1) binding, explore riboswitch's induced fit and/or conformational selection folding mechanism, and study how designed drug compounds regulate the riboswitch folding pathway. Finally, this nanopore molecular machine can be utilized as RNA switch-based biosensor for detection a variety of disease-derived ligands and proteins.

Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A

AbstractIntrinsically disordered proteins have been reported to undergo disorder‐to‐order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C‐terminal half of the protein. Herein, native ion mobility mass spectrometry (IM‐MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. The trimeric Cdk2/cyclin A/p27‐KID complex possesses significant structural heterogeneity compared to Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N‐ and C‐termini of p27 interact with Cdk2/cyclin A in multiple, closely associated states.

Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A.

Intrinsically disordered proteins have been reported to undergo disorder-to-order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C-terminal half of the protein. Herein, native ion mobility mass spectrometry (IM-MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. The trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity compared to Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N- and C-termini of p27 interact with Cdk2/cyclin A in multiple, closely associated states.

Position-, disorder-, and salt-dependent diffusion in binding-coupled-folding of intrinsically disordered proteins.

Successful extensions of protein-folding energy landscape theory to intrinsically disordered proteins' (IDPs') binding-coupled-folding transition can enormously simplify this biomolecular process into diffusion along a limited number of reaction coordinates, and the dynamics subsequently is described by Kramers' rate theory. As the critical pre-factor, the diffusion coefficient D has direct implications on the binding kinetics. Here, we employ a structure-based model (SBM) to calculate D in the binding-folding of an IDP prototype. We identify a strong position-dependent D during binding by applying a reaction coordinate that directly measures the fluctuations in a Cartesian configuration space. Using the malleability of the SBM, we modulate the degree of conformational disorder in an isolated IDP and determine complex effects of intrinsic disorder on D varying for different binding stages. Here, D tends to increase with disorder during initial binding but shows a non-monotonic relationship with disorder in terms of a decrease-followed-by-increase in D during the late binding stage. The salt concentration, which correlates with electrostatic interactions via Debye-Hückel theory in our SBM, also modulates D in a stepwise way. The speeding up of diffusion by electrostatic interactions is observed during the formation of the encounter complex at the beginning of binding, while the last diffusive binding dynamics is hindered by non-native salt bridges. Because D describes the diffusive speed locally, which implicitly reflects the roughness of the energy landscape, we are eventually able to portray the binding energy landscape, including that from IDPs' binding, then to binding with partial folding, and finally to rigid docking, as well as that under different environmental salt concentrations. Our theoretical results provide key mechanistic insights into IDPs' binding-folding, which is internally conformation- and externally salt-controlled with respect to diffusion.

Structural Origins of FRET-Observed Nascent Chain Compaction on the Ribosome.

A fluorescence signal arising from a Förster resonance energy transfer process was used to monitor conformational changes of a domain within the E. coli protein HemK during its synthesis by the ribosome. An increase in fluorescence was observed to begin 10 s after translation was initiated, indicating the domain became more compact in size. Since fluorescence only reports a single value at each time point it contains very little information about the structural ensemble that gives rise to it. Here, we supplement this experimental information with coarse-grained simulations that describe protein conformations and transitions at a spatial resolution of 3.8 Å. We use these simulations to test three hypotheses that might explain the cause of domain compaction: (1) that poor solvent quality conditions drive the unfolded state to compact, (2) that a change in the dimension of the space the domain occupies upon moving outside the exit tunnel causes compaction, or (3) that domain folding causes compaction. We find that domain folding and dimensional collapse are both consistent with the experimental data, while poor-solvent collapse is inconsistent. We identify alternative dye labeling positions on HemK that upon fluorescence can differentiate between the domain folding and dimensional collapse mechanisms. Partial folding of domains has been observed in C-terminally truncated forms of proteins. Therefore, it is likely that the experimentally observed compact state is a partially folded intermediate consisting, according to our simulations, of the first three helices of the HemK N-terminal domain adopting a native, tertiary configuration. With these simulations we also identify the possible cotranslational folding pathways of HemK.

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha.

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

Resolution of Large Outer Retinal Fold Following Retinal Detachment Surgery With Visual Acuity Restoration to 20/20: A Case Report and Literature Review

Purpose: To report a case of macular fold formation following surgical repair of rhegmatogenous retinal detachment in a 57-year-old man treated with pars plana vitrectomy (PPV), subretinal fluid endodrainage, gas-fluid exchange and sulfur hexafluoride tamponade, and postoperative face-down positioning. We also discuss etiology and prevention of macular fold following retinal detachment surgery and review the literature regarding current trends in the management of this complication. Methods: Case report and literature review. Results: The macular fold has resolved spontaneously with eventual flattening of and restoration of vision to 20/20 at 12 months postoperatively. Conclusions: This case and other recently published reports suggest good outcomes with conservative management of outer retinal folds and partial thickness macular folds. Further research is needed to identify clinical and imaging features that could be used to build criteria to guide management of this complication.

Stability of an aggregation-prone partially folded state of human profilin-1 correlates with aggregation propensity

A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of familial forms of ALS (fALS), also known as Lou Gehrig's disease. The pathogenic potential of these mutations is linked to the formation of intracellular inclusions of the mutant proteins and correlates with the mutation-induced destabilization of its native, fully folded state. However, the mechanism by which these mutations promote misfolding and self-assembly is yet unclear. Here, using temperature-jump and stopped-flow kinetic measurements, we show that, during refolding, WT profilin-1 transiently populates a partially folded (PF) state endowed with hydrophobic clusters exposed to the solvent and with no detectable secondary structure. We observed that this conformational state is marginally stable at neutral pH but becomes significantly populated at mildly acidic pH. Interestingly, the fALS-associated mutations did not cause a change in the refolding mechanism of profilin-1, but induced a stabilization of the PF state. In the presence of preformed profilin-1 aggregates, the PF state, unlike the unfolded and folded states, could interact with these aggregates via nonspecific hydrophobic interactions and also increase thioflavin-T fluorescence, revealing its amyloidogenic potential. Moreover, in the variants tested, we found a correlation between conformational stability of PF and aggregation propensity, defining this conformational state as an aggregation-prone folding intermediate. In conclusion, our findings indicate that mutation-induced stabilization of a partially folded state can enhance profilin-1 aggregation and thereby contribute to the pathogenicity of the mutations.

Quantitative Hydroxyl Radical Footprinting Study Reveals Structural Details of the Disorder-to-Order Transition in Amyloid-Beta (1-42) Oligomerization

This work presents a novel integrated analysis approach to characterize the intrinsically disordered 42 amino acid long β-amyloid peptide (Aβ42) and to elucidate mechanisms driving its misfolding and aggregation into neurotoxic oligomers. We describe this approach, which uses synchrotron based hydroxyl radical footprinting (HRF) results to determine residue specific solvent accessibilities for each of seventeen individual amino acids in monomer, multimer (2-4mer), and oligomer (∼150 kDa) Aβ42 sample preparations. Results complement published solid state NMR constraints with solution state structural details for the oligomers, and provide much needed experimental support for molecular dynamics (MD) computational studies of the monomer and 2-4mer species, where solid state NMR cannot be used. The pattern of protection from hydroxyl radical attack in the Aβ42 monomer (relative to the same residues in the fragmented unstructured peptide) shows partial folding in the monomer. The sequence location of this partial folding is in agreement with MD predictions of transient β-strand formation in the hydrophobic core and C-terminal regions. Sequence-specific protection (i.e. reduced side chain solvent accessibility) upon further assembly into the 2-4mer and oligomer species suggest specific conformers that are selected from the intrinsically disordered monomer ensemble to drive oligomerization. Results of the current study are also consistent with general models developed from previously published solid state NMR oligomer studies from this and other laboratories.

Folding Proteins Outside Their Native Environment: Folding the Innards of an Outer Membrane Transporter

During metabolite transport, TonB dependent receptors, found in the outer membranes of many bacterial species, are hypothesized to unfold their N-terminal “plug” domain. The 130 aa plug normally is folded inside a ∼450-550 aa, 22-strand β barrel, and must undergo significant rearrangement to allow passage of large (∼1kDa) substrates such as vitamin B12. In the crystal structures, the interface between the plugs and the inside surface of the barrels is well hydrated and is composed primarily of H-bonded, charged, and polar contacts, despite being located inside the membrane plane. Here, we present CD, NMR, and SAXS results indicating that several such plug domains are unfolded in solution whereas those from BtuB and CirA adopt a partial globular fold consistent with their crystal structures. In the crystal structure, the plugs have internal void volumes, suggesting that they can unfold non-cooperatively, as has been shown in urea denaturation EPR as well as recent AFM experiments. This is supported by our kinetic folding data and pressure denaturation NMR experiments, which has implications for folding pathways as well as the transport function. NMR studies of the BtuB & CirA plug domains will be presented, in which we take a divide and conquer approach to finding the minimal folding subunit of the plug.

Unraveling the differential structural stability and dynamics features of T7 endolysin partially folded conformations

BackgroundCharacterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5–3, T7L PF states display differential ANS binding characteristics. MethodsCD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations. ResultsStructural studies indicated a partial loss of secondary/tertiary structures compared to its native state. The loss in the tertiary structure and the hydrophobic core opening increases upon decrease of pH from 5 to 3. Thermal denaturation experiments delineated that the pH 5 conformation is thermally irreversible in contrast to pH 3, depicting that hydrophobic core opening is essential for thermal reversibility. Further, urea dependent unfolding features of PF state at pH 5 and 4 evidenced for a collapsed conformation at intermediate urea concentrations. Residue level studies revealed that α1-helix and β3-β4 segment of T7L are the major contributors for such a structural collapse and inherent dynamics. ConclusionsThe results suggested that the low pH PF states of T7L are heterogeneous and exhibits differential structural, unfolding, thermal reversibility, and dynamic features. General significanceUnraveling the structure-stability characteristics of different endolysin conformations is essential for designing novel chimeric and engineered phage endolysins as broadband antimicrobial agents over a varied pH range.

Fold Over Plan for Uniform Design

This article discusses the fold over problem for uniform design in terms of the centered L2 discrepancy. Both full fold over plan and partial fold over plan are studied. Illustrative examples are given.