Beta-sheet Secondary Structure Research Articles

FTIR and Femtosecond 2D-IR Spectroscopy of Azidohomoalanine-Labeled PDZ3 from PSD-95: Site-Specific Probing of Ultrafast Dynamics and Electrostatics in Proteins

Vibrational spectroscopy is a highly sensitive tool to study the structure and function of proteins. The absorption frequency of particular functional groups such as azides or nitriles can be sensitive to even small changes in the electrostatic environment. We use the methionine surrogate azidohomoalanine as a spectroscopic probe, which can be incorporated site-selectively during protein synthesis. Here we present recent work on the 3rd PDZ domain of PSD-95, a protein domain of 10.7kDa involved in protein-protein-interaction and signaling processes. The PDZ domain has various features that make it a suitable target for testing the application of an intrinsic azide group as a probe for the electrostatic environment. The protein displays both alpha-helical and beta-sheet secondary structure elements and has a hydrophobic peptide binding pocket. We incorporated azidohomoalanine at six positions in different secondary structures, in the interior and at the surface of the protein, as well as close to the binding pocket. Static IR spectra of all mutants show a clear correlation of the azide absorption frequency with the hydrophobicity of the surrounding side chains, the positions of which are known from x-ray structures. Furthermore changes in hydrophobicity upon ligand binding can be monitored, not only in FTIR data, but also in time-resolved 2D-IR spectroscopy. Using ultrafast 2D-IR spectroscopy we are able to measure fluctuations in the protein environment around our local probe on a picosecond timescale. Our data show that already on this timescale differences in dynamics upon ligand binding are induced.

Natural Biomolecules and Protein Aggregation: Emerging Strategies against Amyloidogenesis

Biomolecular self-assembly is a fundamental process in all organisms. As primary components of the life molecular machinery, proteins have a vast array of resources available to them for self-assembly in a functional structure. Protein self-assembly, however, can also occur in an aberrant way, giving rise to non-native aggregated structures responsible for severe, progressive human diseases that have a serious social impact. Different neurodegenerative disorders, like Huntington’s, Alzheimer’s, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates, generally termed “amyloid,” that share several physicochemical features: a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and protease resistance. Conformational constrains, hydrophobic and stacking interactions can play a key role in the fibrillogenesis process and protein–protein and peptide–peptide interactions—resulting in self-assembly phenomena of peptides yielding fibrils—that can be modulated and influenced by natural biomolecules. Small organic molecules, which possess both hydrophilic and hydrophobic moieties able to bind to peptide/protein molecules through hydrogen bonds and hydrophobic and aromatic interactions, are potential candidates against amyloidogenesis. In this review some significant case examples will be critically discussed.

The Role of Pro, Gly Lys, and Arg Containing Peptides on Amyloid-Beta Aggregation

Genetic, biochemical and pathological evidence support that self-assembly of amyloid-beta (Aβ) peptide into toxic aggregates is implicated as the cause of Alzheimer’s disease. An attractive therapeutic strategy for the treatment of AD is to prevent or interfere with Aβ aggregation. A systematic investigation of the effects of proline-, glycine-, arginine- and lysine- containing peptides (PGKLVYA, KKLVFFARRRRA and KKLVFFA) on the beta-amyloid aggregation was made using FTIR, circular dichroism, ANS binding, ThT binding and TEM analysis. These peptides are based on the central hydrophobic region of Aβ (residues 16–20), which is believed to be crucial in Aβ self-association. There is increasing evidence to suggest that protein aggregation, including amyloid fibril formation results from the strong self-association tendency of the partially folded intermediates. Addition of PGKLVYA and KKLVFFARRRRA resulted in increase in ANS fluorescence intensity, suggesting enhanced exposure of hydrophobic surface area. As observed by ThT and TEM analysis PGKLVYA and KKLVFFARRRRA promote non-fibrillar ensembles, while peptide KKLVFFA accelerated the fibrillization of Aβ peptide by stabilizing intermolecular interactions. Circular dichroism and FTIR data showed that PGKLVYA and KKLVFFARRRRA effectively prevented amyloid-beta (Aβ) peptide adopting the beta-sheet secondary structure correlated with fibrillogenesis. This result indicates that PGKLVYA and KKLVFFARRRRA might have triggered another mechanism of Aβ assembly.

Self-Assembly of PEGylated Peptide Conjugates Containing a Modified Amyloid β-Peptide Fragment

The self-assembly of PEGylated peptides containing a modified sequence from the amyloid beta peptide, FFKLVFF, has been studied in aqueous solution. PEG molar masses PEG1k, PEG2k, and PEG10k were used in the conjugates. It is shown that the three FFKLVFF-PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The beta-sheet secondary structure of the peptide is retained in the FFKLVFF fibril core. At sufficiently high concentrations, FFKLVFF-PEG1k and FFKLVFF-PEG2k form a nematic phase, while PEG10k-FFKLVFF exhibits a hexagonal columnar phase. Simultaneous small angle neutron scattering/shear flow experiments were performed to study the shear flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without disruption of the FFKLVFF beta-sheet structure leading to characteristic peaks in the X-ray diffraction pattern and FTIR spectra. The stability of beta-sheet structures was also studied in blends of FFKLVFF-PEG conjugates with poly(acrylic acid) (PAA). While PEG crystallization is only observed up to 25% PAA content in the blends, the FFKLVFF beta-sheet structure is retained up to 75% PAA.

PB1-F2 Influenza A Virus Protein Adopts a β-Sheet Conformation and Forms Amyloid Fibers in Membrane Environments

The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

Role of Polyalanine Domains in β‐Sheet Formation in Spider Silk Block Copolymers

Genetically engineered spider silk-like block copolymers were studied to determine the influence of polyalanine domain size on secondary structure. The role of polyalanine block distribution on beta-sheet formation was explored using FT-IR and WAXS. The number of polyalanine blocks had a direct effect on the formation of crystalline beta-sheets, reflected in the change in crystallinity index as the blocks of polyalanines increased. WAXS analysis confirmed the crystalline nature of the sample with the largest number of polyalanine blocks. This approach provides a platform for further exploration of the role of specific amino acid chemistries in regulating the assembly of beta-sheet secondary structures, leading to options to regulate material properties through manipulation of this key component in spider silks.

Copper abolishes the beta-sheet secondary structure of preformed amyloid fibrils of amyloid-beta(42).

The observation of the co-deposition of metals and amyloid-beta(42) (Abeta(42)) in brain tissue in Alzheimer's disease prompted myriad investigations into the role played by metals in the precipitation of this peptide. Copper is bound by monomeric Abeta(12) and upon precipitation of the copper-peptide complex thereby prevents Abeta(42) from adopting a beta-sheet secondary structure. Copper is also bound by beta-sheet conformers of Abeta(42), and herein we have investigated how this interaction affects the conformation of the precipitated peptide. Copper significantly reduced the thioflavin T fluorescence of aged, fibrillar Abeta(42) with, for example, a 20-fold excess of the metal resulting in a ca 90% reduction in thioflavin T fluorescence. Transmission electron microscopy showed that copper significantly reduced the quantities of amyloid fibrils while Congo red staining and polarized light demonstrated a copper-induced abolition of apple-green birefringence. Microscopy under cross-polarized light also revealed the first observation of spherulites of Abeta(42). The size and appearance of these amyloid structures were found to be very similar to spherulites identified in Alzheimer's disease tissue. The combined results of these complementary methods strongly suggested that copper abolished the beta-sheet secondary structure of pre-formed, aged amyloid fibrils of Abeta(42). Copper may protect against the presence of beta-sheets of Abeta(42) in vivo, and its binding by fibrillar Abeta(42) could have implications for Alzheimer's disease therapy.

Shear flow promotes amyloid- fibrilization

The rate of formation of amyloid fibrils in an aqueous solution of amyloid-beta (Abeta) is greatly increased when the solution is sheared. When Abeta solution is stirred with a magnetic stirrer bar at 37 degrees C, a rapid increase in thioflavin T fluorescence is observed. Atomic Force Microscopy (AFM) images show the formation of aggregates, the growth of fibrils and the intertwining of the fibrils with time. Circular dichroism (CD) spectroscopy of samples taken after stirring shows a transition from random coil to alpha-helix to beta-sheet secondary structure over 20 h at 37 degrees C. The fluorescence, AFM and CD measurements are all consistent with the formation of amyloid fibrils. Quiescent, non-stirred solutions incubated at 37 degrees C showed no evidence of amyloid formation over a period of 3 days. Couette flow was found to accelerate the formation of amyloid fibrils demonstrating that the primary effect of stirring is not mixing but shearing. Only very small shear forces are applied to individual molecules in our experiments. Simple calculation suggests that the force is too small to support a hypothesis that shearing promotes partial unfolding of the protein as is observed.

Macrocyclic Design Strategies for Small, Stable Parallel β-Sheet Scaffolds

Pairs of short peptide strands can be induced to adopt an antiparallel beta-sheet secondary structure in aqueous solution via a macrocyclic constraint, as illustrated by many natural and designed peptides. We show that an analogous strategy is successful for creation of small units of parallel beta-sheet secondary structure in aqueous solution. Cyclization in this case requires nonpeptide segments for N-to-N and C-to-C interstrand linkage. Surprisingly, we find that only one of these segments needs to be preorganized.

Characterization of the Boundary Zone of a Cast Protein Drop: Fibroin β-Sheet and Nanofibril Formation

We have studied a cast fibroin drop with grazing incidence small-angle X-ray scattering, imaging, and spectroscopy techniques. Optical microscopy shows that the dried drop forms a boundary zone. Grazing-incidence small-angle X-ray scattering performed with a synchrotron radiation microbeam in the boundary zone suggests the formation of nanometer-sized domains with one-dimensional paracrystalline order. Atomic force microscopy and scanning electron microscopy support a nanofibrillar morphology. MicroRaman spectroscopy shows the formation of beta-sheet secondary structure in the boundary zone, which is attributed to a shearing effect due to the retraction of the drop boundary upon evaporation.

Intra-protein hydrogen bonding is dynamically stabilized by electronic polarization

Molecular dynamics (MD) simulation has been carried out to study dynamical stability of intra-protein hydrogen bonds based on two set of atomic charges, the standard AMBER charge and the polarized protein-specific charge (PPC). The latter is derived from quantum mechanical calculation for protein in solution using a recently developed molecular fractionation with conjugate caps-Poisson-Boltzmann (MFCC-PB) approach and therefore includes electronic polarization effect of the protein at native structure. MD simulations are performed for a number of benchmark proteins containing helix and/or beta sheet secondary structures. The computational result shows that occupancy percentage of hydrogen bonds averaged over simulation time, as well as the number of hydrogen bonds as a function of simulation time, is consistently higher under PPC than AMBER charge. In particular, some intra-protein hydrogen bonds are found broken during MD simulation using AMBER charge but they are stable using PPC. The breaking of some intra-protein hydrogen bonds in AMBER simulation is responsible for deformation or denaturing of some local structures of proteins during MD simulation. The current study provides strong evidence that hydrogen bonding is dynamically more stable using PPC than AMBER charge, highlighting the stabilizing effect of electronic polarization on protein structure.

Different Individual Amyloid Fibrils Exhibit Different Beta Sheet Secondary Structures via Near-field Infrared Spectroscopy

Morphology and secondary structure of individual amyloid fibrils synthesized from the #21-31 peptide fragment of B2-microglobulin were obtained using apertureless near-field scanning infrared microscopy (ANSIM). The sample exhibits a heterogeneous population of fibrils: some fibrils exhibit parallel and some with anti-parallel beta sheet structures. ANSIM is an optical technique which uses a carbon monoxide IR laser coupled with a tapping mode atomic force microscope using homodyne detection demodulated at the tip frequency. The experimental near-field spectra correlate strongly to the calculated near-field spectrum as well as the far-field spectrum. Near-field images exhibit high attenuation of the amyloid fibrils at approximately 1630 cm−1. Strong attenuation of the incident IR radiation at 1691 cm−1 corresponding to the presence of anti-parallel beta sheet structure is observed for a fraction of the fibrils. The observation of the anti-parallel beta sheet conformation is linked to the synthesis method used to produce these amyloid fibrils. It is shown that the addition of trimethylamine N-oxide (TMAO) can accelerate the formation of amyloid fibrils and induce anti-parallel beta sheet structure.

Amphiphilic Peptides That Self Assemble into Nanomicelles and Vesicles

Self-Assembling peptide nanovesicles are attractive candidates for drug delivery. Here we report the characterization of specific amphiphilic peptides (up to 23-amino acids in length) that undergo supramolecular assembly to form both mono- and hetero-assemblies that form nanomicelles and larger vesicles (150nm) in aqueous solutions. Depending on the peptide's composition and the pH of the aqueous medium during assembly, the peptides adopt either a micellar or vesicular structure. Simple amphipathic sequences adopt a micellar structure (<50nm) at low to neutral pH. When pairs of lyophilized peptides with different lengths are co-dissolved in an unbuffered Carboxyfluorescein solution at a 1:1 molar ratio, they self-assemble into small vesicles that are visualized using a confocal microscope. These intact vesicles average about 150nm in size (as determined by confocal images) and are capable of entrapping solutes. CD and FTIR analyses of such mixtures indicated a tendency of the peptide to adopt a beta sheet secondary structure. Isothermal Titration Calorimetry (ITC) revealed the Critical Aggregation Concentration (CAC) for the individual peptides to be less than 1mM, which is a useful property during drug delivery. These nanostructures can be used as models for further developments.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mechanism of zinc(II)-promoted amyloid formation: zinc(II) binding facilitates the transition from the partially α-helical conformer to aggregates of amyloid β protein(1–28)

The amyloidoses are a group of disorders characterized by aberrant protein folding and assembly, leading to the deposition of insoluble protein fibrils (amyloid), which provokes cell dysfunction and later cell death. One of the physiologically relevant environmental factors able to affect the conformation and hence the aggregation properties of amyloidogenic proteins/peptides is metal ions. Zn(II) promotes aggregation of most amyloidogenic peptides/proteins in vitro, including amyloid beta protein (Abeta), but the underlying mechanism is not known. To better understand this mechanism the present study focused on the partially alpha-helical conformer, supposed to be an intermediate in Abeta aggregation. This partially alpha-helical conformer is stabilized by 10-20% 2,2,2-trifluoroethanol (TFE): therefore, the influence of Zn binding on the aggregation of the amylidogenic model peptide Abeta(1-28) (Abeta28) was investigated at different TFE concentrations. The results showed a synergistic effect of Zn(II) and 10% TFE, i.e., that either Zn or 10% TFE accelerated Abeta28 aggregation on its own, but with them together an at least 10 times promotion of Abeta28 aggregation was observed. Further studies by thioflavin T fluorescence spectroscopy, transmission electron microscopy, and circular dichroism (CD) spectroscopy suggested that the aggregates of Zn-Abeta28 formed in 10%TFE contain a beta-sheet secondary structure and are more of the amyloid type. CD spectroscopy indicated that Zn binding disrupted partially the alpha-helical structure of Abeta28 in TFE. Thus, we propose that the promotion of Abeta28 aggregation by Zn is based on the transformation of the partially alpha-helical conformer (intermediate) towards the beta-sheet amyloid structure by a destabilization of the alpha-helix in the intermediate.

Characterization of Fluoroalcohols-Induced Intermediates of Mucor miehei Lipase at Low pH

We have previously characterized an acid-unfolded (U(A)) state of Mucor miehei lipase at pH 2. The effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) resulted in characterization of molten-globule (MG) like states with beta-sheet secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. alpha-Helical states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.

Tracking Fiber Formation in Human Islet Amyloid Polypeptide with Automated 2D-IR Spectroscopy

Amyloid forming proteins have been implicated in many human diseases. The kinetics of amyloid fiber formation are of particular interest because evidence points to intermediate folding structures as potential cytotoxic species. The standard methods for monitoring the kinetics are to use fluorescence or circular dichroism spectroscopy, which do not uniquely resolve secondary structures. In this work, we use a new technology for rapidly scanning 2D-IR spectra that allows us to follow the fiber formation kinetics of the human islet amyloid polypeptide (hIAPP) that is involved in type II diabetes. Spectroscopic markers are identified that uniquely monitor random coil versus beta-sheet secondary structures as well as probe beta-sheet elongation and stacking. Our measurements provide more rigorous kinetics for the secondary structure evolution of amyloid formation than is available with other techniques.

Full‐length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathways

As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes beta-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, Agg(HP), which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, Agg(HP) transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially beta-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, I(HP), is formed under high pressure. After pressure release, I(HP) relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, I(HP), constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.

De Novo Design of Strand-Swapped β-Hairpin Hydrogels

De novo designed peptides, capable of undergoing a thermally triggered beta-strand-swapped self-assembly event leading to hydrogel formation were prepared. Strand-swapping peptide 1 (SSP1) incorporates an exchangeable beta-strand domain composed of eight residues appended to a nonexchangeable beta-hairpin domain. CD shows that, at pH 9 and temperatures less than 35 degrees C, this peptide adopts a random coil conformation, rendering it soluble in aqueous solution. On heating to 37 degrees C or greater, SSP1 adopts a beta-hairpin that displays an exchangeable beta-strand region. The exchangeable strand domain participates in swapping with the exchangeable domain of another peptide, affording a strand-swapped dimer. These dimers further assemble into fibrils that define the hydrogel. A second peptide (SSP2) containing an exchangeable strand composed of only four residues was also studied. Microscopy and scattering data show that the length of the exchangeable domain directly influences the fibril nanostructure and can be used as a design element to construct either twisted (SSP1) or nontwisted (SSP2) fibril morphologies. CD, FTIR, and WAXS confirm that both peptides adopt beta-sheet secondary structure when assembled into fibrils. Fibril dimensions, as measured by TEM, AFM, and SANS indicate a fibril diameter of 6.4 nm, a height of 6.0 nm, and a pitch of 50.4 nm for the twisted SSP1 fibrils. The nontwisted SSP2 fibrils are 6.2 nm in diameter and 2.5 nm in height. Oscillatory rheology, used to measure bulk hydrogel rigidity, showed that the gel composed of the nontwisted fibrils is more mechanically rigid (517 Pa at 6 rad/s) than the gel composed of twisted fibrils (367 Pa at 6 rad/s). This work demonstrates that beta-strand-swapping can be used to fabricate biomaterials with tunable fibril nanostructure and bulk hydrogel rheological properties.

Stabilization of the N‐terminal β‐hairpin of ubiquitin by a terminal hydrophobic cluster

Study of model beta-hairpin peptides allows for better understanding of the factors involved in the formation of beta-sheet secondary structure in proteins. It is known that turn sequence, sidechain-sidechain interactions, interstrand hydrogen bonding, and beta-sheet propensity of residues are all important for beta-hairpin stability in aqueous solution. However, interactions of the sidechains of the terminal residues of hairpins are thought to contribute little to overall hairpin stability since these residues are typically frayed. Here, the authors report a stabilizing hydrophobic cluster of residues at the termini of the naturally occurring excised N-terminal beta-hairpin of Ubiquitin that folds autonomously in aqueous solution. Our data show that deletion of Met1 and Val17 from this hairpin destabilized the folded state in both aqueous solution and in aqueous-methanol solutions. These results suggest that interactions of terminal residues which are usually frayed can nonetheless contribute significantly to overall stability of beta-hairpin.

Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions.

Computational design of surface charge-charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, DeltaT (m) = 12.3 +/- 0.2 degrees C, and stability, DeltaDeltaG(25 degrees C) = 7.1 +/- 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge-charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.