Β-sheet Aggregates Research Articles

G.P.62: Molecular cell stress mechanisms in an in vitro model of IBM

Inclusion body myositis (IBM) is the most common acquired myopathy in patients over 50years. The pathology involves an inflammatory response and β-amyloid deposits in muscle fibers. It is believed that MAP kinases such as ERK, JNK and p38 signaling pathways mediate the inflammatory signaling in cells. There is evidence that autophagic activity plays a crucial role in the pathogenesis of IBM. Using an in vitro model of IBM, transcription factors, such as MAP kinases, the autophagic pathway and accumulation of β-amyloid were examined. Rhabdomyosarcoma cells were exposed to the cytokines IFN-γ and IL-1β for 24 and 48h. In addition, the cells were treated with various MAP kinase inhibitors. The protein expression of IL-1β, CXCL9 and of phosphorylated and non-phosphorylated forms of the different MAP kinases was determined by western blotting. The effects on autophagy were analyzed using stably transfected GFP-LC3-rhabdomyosarcoma cells. β-Sheet aggregates were visualized by immunocytochemistry. Cell death was determined via propidium iodide staining. Stimulation of muscle cells with IL-1β and IFN-γ led to an increased phosphorylation of ERK, but neither JNK nor p38. The ERK inhibitor PD98059 diminished the expression of pro-inflammatory markers as well as the accumulation of β-amyloid. Immunocytochemical analysis of GFP-LC3 muscle cells showed that IL-1β and IFN-γ led to an increase of autophagic activity, upregulation of APP and subsequent accumulation of β-sheet aggregates. Taken together, these data demonstrate that MAP kinases, such as ERK are of importance for the formation of β-amyloid and the regulation of autophagic activity in an in vitro model of IBM, especially under conditions of proinflammatory cell stress. Thus, ERK could provide an important link between inflammation and protein deposition. This study contributes to a better understanding of the pathogenesis of chronic muscle inflammation as in IBM.

Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein.

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the β-sheet propensities of the native sequences.Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks”) promote surface activity by mimicking the α-helix and membrane topography of native SP-C.Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu−–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency.Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with ‘amyloid-like’ properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard 12C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu−–Lys+ insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu−–Lys+ ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C.Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.

Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

Functional protein from cumin seed (Cuminum cyminum): Optimization and characterization studies

Cumin seed protein isolate (CSPI) was found to be a potential source of bioactive protein. Box-Behnken Design was used in order to optimize the protein extraction parameters: extraction time (X1: 0.5–1.5 h), extraction temperature (X2: 20–40 °C) and buffer-to-sample ratio (X3: 10–30 mL/g). Extraction conditions for maximum protein yield were corresponding to X1 = 0.6 h, X2 = 26.3 °C and X3 = 10 mL/g. A close agreement between experimental (44.98 mg/g) and predicted (45.19 mg/g) values was found. The physicochemical properties (i.e. amino acid composition, protein components, protein structure) and its potential bioactivities (i.e. antioxidant and antidiabetic activity) were also evaluated, giving a better understanding of general structure and properties of CSPI. 2S albumin, 7S globulin, 11S globulin and lectin were found as the components of the extracted seed protein. Structure of CSPI was a mixture of intramolecular β-sheet (1639 cm−1), random coil (1642 cm−1), α-helix (1655 cm−1), β-turns (1660 cm−1) and antiparallel β-sheet aggregates (1690 cm−1) as shown in the FTIR spectra. CSPI was predominant with the Tyr, Glu, Asp, Arg, Leu and Phe, which could be considered as a high quality of natural protein. In addition, CSPI showed appreciable DPPH free radical scavenging activity (47.7 %DPPHsc/μg) and reducing power (12.4 mM/μg), which implied that CSPI could be used as a natural antioxidant agent. However, CSPI showed a relatively low α-amylase inhibition activity (6.7%). These findings demonstrated that CSPI could be a used as a potential nutraceutical or ingredient of functional and health-promoting foods.

Rationally Designed Peptoids Modulate Aggregation of Amyloid-Beta 40

Alzheimer's disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States. Plaques composed of aggregated amyloid-beta protein (Aβ) accumulate between the neural cells in the brain and are associated with dementia and cellular death. Many strategies have been investigated to prevent Aβ self-assembly into disease-associated β-sheet amyloid aggregates; however, a promising therapeutic has not yet been identified. In this study, a peptoid-based mimic of the peptide KLVFF (residues 16-20 of Aβ) was tested for its ability to modulate Aβ aggregation. Peptoid JPT1 includes chiral, aromatic side chains to induce formation of a stable helical secondary structure that allows for greater interaction between the aromatic side chains and the cross β-sheet of Aβ. JPT1 was found to modulate Aβ40 aggregation, specifically decreasing lag time to β-sheet aggregate formation as well as the total number of fibrillar, β-sheet structured aggregates formed. These results suggest that peptoids may be able to limit the formation of Aβ aggregates that are associated with AD.

Fibril formation from pea protein and subsequent gel formation.

The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

Inhibition of Fibril Formation by Tyrosine Modification of Diphenylalanine: Crystallographic Insights

The self-assemblies of diphenylalanine and its tyrosine analogues have been investigated. The peptide Boc-Phe-Phe-OMe (1), having a sequence identity with the central hydrophobic cluster (CHC) of Alzheimer’s β-amyloid diphenylalanine motif, self-assembles to produce twisted fibrils. In contrast, the tyrosine-modified analogues Boc-Phe-Tyr-OMe (2), Boc-Tyr-Phe-OMe (3), and Boc-Tyr-Tyr-OMe (4), self-assemble to form microspheres. The X-ray crystallography reveal that the peptide 1 adopts an inverse γ-turn structure and self-associates as a hydrogen-bonded chain of molecules along a 2-fold screw axis, whereas the tyrosine-modified analogues exhibit parallel β-sheet aggregation and cyclic packing in higher-order assembly. The structural analysis of the peptides as described here can serve as a basis for de novo design and therapeutics.

Aggregation Gatekeeper and Controlled Assembly of Trpzip β-Hairpins

Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well-studied β-hairpins, Trpzip1 and Trpzip2. Previous studies suggested that Trpzip2 remains monomeric up to a concentration of ∼15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides differ only in their turn sequences (i.e., GN vs NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, because of a more favorable orientation. In support of this hypothesis, we find that increasing the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analogue, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys side chain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel β-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel β-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.

Blockade of the formation of insoluble ubiquitinated protein aggregates by EGCG3"Me in the alloxan-induced diabetic kidney.

BackgroundRenal accumulation of reactive carbonyl compounds (RCCs) has been linked to the progression of diabetic nephropathy. We previously demonstrated that carbonyl stress induces the formation of amino-carbonyl cross-links and sharply increases the content of β-sheet-rich structures, which is the seed of insoluble aggregates formation, and tea catechin (-)-epigallocatechin 3-gallate (EGCG) can reverse this process in vitro and in vivo. In this study, methylated derivative (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3”Me) was hypothesized to neutralize carbonyl stress mediating the formation of insoluble ubiquitinated protein (IUP) aggregates, and reduce the early development of diabetic nephropathy.Methods and resultsDiabetes was induced in mice by intraperitoneally injecting alloxan monohydrate (200 mg/kg/d) twice and administering EGCG3”Me by gavage for 15 d. Reagent case and western blot results showed that, in diabetic kidneys, the carbonyl proteins in the serum increased; and in insoluble protein fraction, 4-hydroxynonenal-modified proteins, IUP aggregates and p62 accumulated; FT-IR study demonstrated that the lipid content, anti-parallel β-sheet structure and aggregates increased. EGCG3”Me treatment could effectively reverse this process, even better than the negative control treatment.ConclusionsEGCG3”Me exhibiting anti-β-sheet-rich IUP aggregate properties, maybe represents a new strategy to impede the progression of diabetic nephropathy and other diabetic complications.

Parallel β-Sheet Fibril and Antiparallel β-Sheet Oligomer: New Insights into Amyloid Formation of Hen Egg White Lysozyme under Heat and Acidic Condition from FTIR Spectroscopy

Hen egg white lysozyme (HEWL) is widely used as a model protein for amyloid research. In this study, we aim to use Fourier transform infrared (FTIR) spectroscopy to gain new structural insights into amyloid formation of HEWL under heat and acidic condition. We reveal that the fibril-forming solution of HEWL has the capability to form fibril and oligomer with distinct β-sheet configurations under different temperatures. Amyloid fibril with parallel β-sheet configuration is formed at elevated temperature, while oligomer with antiparallel β-sheet configuration is formed at room temperature. The interplay between fibrillation and oligomerization suggests that the two β-sheet aggregates consume the same amyloidogenic materials such as peptide fragments and nicked HEWL due to lysozyme hydrolysis under heat and acidic condition. Temperature-dependent FTIR reveals that the oligomer is unstable at elevated temperature, demonstrating its off-pathway nature. The temperature-dependent formation of parallel and antiparallel β-sheet configurations discovered in lysozyme system is compared with that of amyloid-β and α-synuclein systems and the implication is discussed.

Vibrational Circular Dichroism Spectra of Lysozyme Solutions: Solvent Effects on Thermal Denaturation Processes

Vibrational spectroscopy has been applied to the study of conformational variation of lysozyme during thermal denaturation. An infrared and vibrational circular dichroism (VCD) analysis of lysozyme in D2O, D2O/EtOD, and D2O/DMSO at different solvent compositions and pH's was accomplished. Complete deuteration effects on amidic groups were revealed through the analysis of the amide I band of lysozyme dissolved in deuterated water. The comparison of IR absorption and VCD spectra of lysozyme in D2O revealed the higher sensitivity of the VCD technique to the presence of partially or fully deuterated secondary structure of lysozyme. Aggregation was induced by thermalization of lysozyme at different solvent compositions and pH values. Interestingly, IR and VCD showed different sensitivities to reveal intermolecular β-sheet aggregation under thermal denaturation. The VCD intensity enhancement was observed as the aggregate dimension increased, whereas IR was sensitive to the nucleation step of the process. Moreover, chirality of supramolecular species was revealed through the analysis of the VCD sign pattern. For the first time, we demonstrate how aggregates produced under different solvent compositions but similar pH's showed the same VCD sign pattern and consequently the same sense of growth.

Potassium and ionic strength effects on the conformational and thermal stability of two aldehyde dehydrogenases reveal structural and functional roles of K⁺-binding sites.

Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K+-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K+-dependent and K+-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K+ ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K+ ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA+) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent T m values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased β-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of β-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.

Metal array fabrication based on ultrasound-induced self-assembly of metalated dipeptides

Pd- and Pt-bound bis-metalated peptides were synthesised by the condensation of Pd- or Pt-aldimine-complex-bound glutamic acids to afford the four possible metal isomers of bis-Pd and bis-Pt-homometalated dipeptides and PdPt- and PtPd-heterometalated dipeptides without metal disproportionation. Ultrasound-induced self-assembly of these bis-metalated peptides proceeded effectively to afford supramolecular gels that displayed well-ordered metal arrays. The formation of parallel β-sheet type aggregates through interpeptide amide-amide hydrogen bonding was confirmed by IR, scanning electron microscopy (SEM), and synchrotron X-ray diffraction analyses (WAXS and SAXS). The mechanism of the ultrasound-induced self-assembly of the metalated dipeptides was elucidated via kinetic and association experiments by (1)H NMR, in which ultrasound-triggered dissociation of intramolecular hydrogen bonds between the chloride ligands of the Pd- and Pt-complexes and amides initially occurred. This was followed by the formation of intermolecular amide-amide hydrogen bonds, which afforded the corresponding oligomeric peptide self-assembly as the nucleus for supramolecular aggregation. The observed first-order relationship of the gelation rate versus the sonication frequency suggested that the microcavitation generated under sonication conditions acted as a crucial trigger and provided a reaction field for efficient self-assembly.

Investigating the Self-Aggregation of a Polyalanine Peptide: Kinetics and Isotope Edited Studies

Exploring the extended β-sheet structures associated with protein aggregation is pivotal for a clear understanding of the factors governing fibril formation. We have recently identified three short alanine based peptides with unusual capabilities to form amyloid-like fibrils in solution, namely Ac-AAAAKAAY-NH2 (AKY8) and (AAKA)4 and (AARA)4. Oligoalanine peptides of these sizes typically exhibit either a statistical coil structure in aqueous solution, or if long enough, α-helical conformations. The fibril formation of AKY8 can be monitored by a strongly enhanced amide I’ Vibrational Circular Dichroism (VCD) signal, which we have shown is due to helical twisting of stacked β-sheets. Due to the inability of the non-tyrosine variant of AKY8, (i.e. AAAAKAA) to aggregate, we hypothesize that the self-aggregation of AKY8 is promoted by cation-π interaction between lysine and tyrosine side-chains. In order to further examine this aggregation mechanism we use Isotope-Edited Infrared (IR) and Vibrational Circular Dichroism Spectroscopy (VCD), which provide site-specific structural information and intra-stand registry of the peptide. First results indicate that there is indeed an in-register arrangement of parallel β-strands in AKY8 with lysine and tyrosine residues aligned between stacks. We also investigate the surprising aggregation of the amphiphilic and positively charged (AAKA)4 peptide which aggregates and forms hydrogels at millimolar concentrations and acidic pH. Interestingly, kinetic studies on this peptide indicate that β-sheet aggregation lacks a nucleation phase. At low concentrations, these peptides decay into disordered structures, according to ECD and IR spectra. At higher peptide concentrations however, this decay is not observed, while some relaxation is still operative, possibly due to a rearrangement of the sheet-like structure. We find that the arginine variant of this peptide, i.e. (AARA)4, also aggregates to form hydrogels, however, this peptide has a long lag phase.

Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-β-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-β, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-β, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed β-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.

The dynamic nature of incubation solution after cooling to room temperature in amyloid formation of hen egg white lysozyme: An FTIR assessment

Amyloid formation of hen egg white lysozyme (HEWL) usually requires elevated temperature, while biophysical characterizations on the incubation solution are often performed at room temperature. Whether maintaining the incubation solution at room temperature results in further structural changes is a significantly important issue that has never been explored. Herein, we use FTIR spectroscopy to assess this issue and reveal that the hot incubation solution of HEWL after cooling to room temperature is in a dynamically evolving state and forms β-sheet aggregates continuously over time. Combined with AFM, we show that these aggregates are non-fibrillar β-sheet aggregates and have vibrational signature distinct from that of fibrillar aggregates. Using FTIR difference spectroscopy, we demonstrate that these non-fibrillar aggregates are in an anti-parallel β-sheet configuration. We also provide a detailed discussion on the spectral assignments for protein aggregates in anti-parallel and parallel β-sheet configurations. With FTIR second derivative technique, we show that these non-fibrillar aggregates are in fact present along with fibrillar aggregates during incubation under elevated temperature but are less stable compared with that at room temperature. Implications from the current work are discussed.

Rubber Elongation Factor (REF), a Major Allergen Component in Hevea brasiliensis Latex Has Amyloid Properties

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

Tryptophan within basic peptide sequences triggers glycosaminoglycan‐dependent endocytosis

Deciphering the structural requirements and mechanisms for internalization of cell-penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase-contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly β-strand structure in complex with HI and form large, stable β-sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.

UV resonance Raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization.

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

PH‐Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins-spidroins-involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild-type (wt) E. australis NT at pH7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5-6Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH7 and pH6 and indicate that the biological function of NT depends on conversion between the two conformations.