Interaction of reduced lysozyme with surfactants. Disulfide effects on reformed structure in micelles

The reformation of secondary structure for unfolded, disulfide reduced hen egg white lysozyme (HEWL) upon interaction with surfactants was studied using CD, fluorescence and IR (infrared) techniques. Equilibrium CD studies showed that reduced HEWL when mixed with negatively charged surfactants, such as SDS (sodium dodecyl sulfate), gradually regains average helical structure to a level equivalent to that obtained for the oxidized form also in SDS, but both forms lose tertiary structure in such environments. This non-native structure recovery process begins with monomer surfactant interaction but at higher concentrations is in part dependent on micelle formation, with the helical fraction reaching its maximum value with each surfactant only above the CMC. Fluorescence changes were more complex, evidencing an intermediate state at lower surfactant concentration. With positively charged surfactants the degree of helicity recovered was less, and the intermediate state in fluorescence was not seen. Stopped flow dynamics studies showed the CD kinetics fit to two exponentials as did the fluorescence. The faster steps in CD and fluorescence detected kinetics appear to be correlated which suggests formation of an intermediate on rapid interaction of the micelle and protein. The second step then reflected attainment of a stable surfactant solvated state which attains maximum helicity and moves the Trps to a more hydrophobic environment, which may occur in independent steps, as the slower kinetics are not well correlated

Equilibrium and Dynamic Spectroscopic Studies of the Interaction of Monomeric β‑Lactoglobulin with Lipid Vesicles at Low pH

β-Lactoglobulin (βLG) is a member of the lipocalin protein family that changes structure upon interacting with anionic surfactants and lipid vesicles under higher-pH conditions at which βLG is dimeric. In this study, a β-sheet to α-helix transformation was also observed for monomeric βLG obtained at pH 2.6 when it was mixed with small unilamellar vesicles (SUVs) of zwitterionic lipids, but being mixed with anionic lipids produced little change. The dynamics and extent of this change were quite dependent on the lipid character, phase, and vesicle size. With 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), at ∼50 °C and pH 2.6, the βLG converted to a substantially helical form upon addition of ∼10 mM lipid in a two-step kinetic process having time constants of ∼1 and ∼25 h, as monitored by circular dichroism (CD). Fluorescence changes were simpler but implied a rapid initial change in the Trp environments followed by a slower process paralleling the change in secondary structure. Polarization attenuated total reflectance Fourier transform infrared results indicate the formed helices are at least partially inserted into the lipid bilayer and the sheet segments are on the surface. Thermal behavior showed that the secondary structure of the lipid-bound βLG had two phases, the first being characteristic of the protein–lipid vesicle interaction and the second following the DSPC phase change after which the protein apparently dissociated from the vesicle. Large unilamellar vesicles had a weaker interaction, as judged by CD, which may correlate to the partial exposure of the hydrophobic parts of the SUV bilayer. Other zwitterionic lipids bound βLG with much slower kinetics and often required sonication to induce interaction, but these also showed dissociation upon lipid phase change. These thermal and kinetic behaviors suggest a mechanism for the interaction of monomeric βLG with zwitterionic lipids different from that seen previously for the dimeric form

Role of Side Chains in β‑Sheet Self-Assembly into Peptide Fibrils. IR and VCD Spectroscopic Studies of Glutamic Acid-Containing Peptides

Poly­(glutamic acid) at low pH self-assembles after incubation at higher temperature into fibrils composed of antiparallel sheets that are stacked in a β₂-type structure whose amide carbonyls have bifurcated H-bonds involving the side chains from the next sheet. Oligomers of Glu can also form such structures, and isotope labeling has provided insight into their out-of-register antiparallel structure [Biomacromolecules 2013, 14, 3880−3891]. In this paper we report IR and VCD spectra and transmission electron micrograph (TEM) images for a series of alternately sequenced oligomers, Lys-(Aaa-Glu)₅-Lys-NH₂, where Aaa was varied over a variety of polar, aliphatic, or aromatic residues. Their spectral and TEM data show that these oligopeptides self-assemble into different structures, both local and morphological, that are dependent on both the nature of the Aaa side chains and growth conditions employed. Such alternate peptides substituted with small or polar residues, Ala and Thr, do not yield fibrils; but with β-branched aliphatic residues, Val and Ile, that could potentially pack with Glu side chains, these oligopeptides do show evidence of β₂-stacking. By contrast, for Leu, with longer side chains, only β₁-stacking is seen while with even larger Phe side chains, either β-form can be detected separately, depending on preparation conditions. These structures are dependent on high temperature incubation after reducing the pH and in some cases after sonication of initial fibril forms and reincubation. Some of these fibrillar peptides, but not all, show enhanced VCD, which can offer evidence for formation of long, multistrand, often twisted structures. Substitution of Glu with residues having selected side chains yields a variety of morphologies, leading to both β₁- and β₂-structures, that overall suggests two different packing modes for the hydrophobic side chains depending on size and type

Arrangement of Fibril Side Chains Studied by Molecular Dynamics and Simulated Infrared and Vibrational Circular Dichroism Spectra

Highly ordered assemblies of β-sheet-forming peptide and protein fibrils have been the focus of much attention because of their multiple and partially unknown biological functions, in particular as related to degenerative neuronal disorders. Recently, vibrational circular dichroism (VCD) spectra have been shown to provide a unique means of detection for such extended structures utilizing modes of the peptide main chain backbone. In the case of poly-glutamic acid, surprising VCD responses were also found for side chain modes. In this study, in an attempt to explain this latter observation and obtain a link between fibrillar structure and its optical spectral properties, molecular dynamics (MD) methods are used to model the geometry and dynamics of assemblies containing repeating β-strands of Glu_<i>n</i>. A crystal-like model was adopted for the MD structure simulations. Infrared and VCD spectra for segments of MD modeled fibrillar geometries were first calculated using density functional theory (DFT), and then, those parameters were applied to larger structures by means of Cartesian coordinate transfer (CCT) of atomic tensors from the segments. The computations suggest the side chains exhibit residual conformational constraints, resulting in local coupling giving rise to non-negligible VCD intensity, albeit with an overall broad distribution. Calculated spectral distributions are qualitatively consistent with the experimental results but do differ in magnitude. The possibility of realistic modeling of vibrational spectra significantly broadens the potential for application of optical spectroscopies in structural studies of these aggregated biopolymers

Dimethyl Sulfoxide Induced Destabilization and Disassembly of Various Structural Variants of Insulin Fibrils Monitored by Vibrational Circular Dichroism

Dimethyl sulfoxide (DMSO) induced destabilization of insulin fibrils has been previously studied by Fourier transform infrared spectroscopy and interpreted in terms of secondary structural changes. The variation of this process for fibrils with different types of higher-order morphological structures remained unclear. Here, we utilize vibrational circular dichroism (VCD), which has been reported to provide a useful biophysical probe of the supramolecular chirality of amyloid fibrils, to characterize changes in the macroscopic chirality following DMSO-induced disassembly for two types of insulin fibrils formed under different conditions, at different reduced pH values with and without added salt and agitation. We confirm that very high concentrations of DMSO can disaggregate both types of insulin fibrils, which initially maintained a β-sheet conformation and eventually changed their secondary structure to a disordered form. The two types responded to varying concentrations of DMSO, and disaggregation followed different mechanisms. Interconversion of specific insulin fibril morphological types also occurred during the destabilization process as monitored by VCD. With transmission electron microscopy, we were able to correlate the changes in VCD sign patterns to alteration of morphology of the insulin fibrils

Effect of Hydrophobic Interactions on the Folding Mechanism of β‑Hairpins

Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTW­ENGKW­TWK-NH2) that is stabilized by two cross-strand Trp–Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process

Insight into the Packing Pattern of β₂ Fibrils: A Model Study of Glutamic Acid Rich Oligomers with ¹³C Isotopic Edited Vibrational Spectroscopy

Polyglutamic acid at low pH forms aggregates and self-assembles into a spiral, fibril-like superstructure formed as a β₂-type sheet conformation that has a more compact intersheet packing than commonly found. This is stabilized by three-centered bifurcated hydrogen bonding of the amide carbonyl involving the protonated glutamic acid side chain. We report vibrational spectroscopic results and analyses for oligopeptides rich in glutamic acid enhanced with ¹³C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and β₂ aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional β₁-sheet aggregates by rapid precipitation from the residual peptides in the higher pH supernatant. By comparing the isotope-enhanced IR and VCD spectra with theoretical predictions, we deduced that the oligo-Glu β₂ structure is based on stacked, twisted, antiparallel β-sheets. The best fit to theoretical predictions was obtained for the strands being out of register, sequentially stepped by one residue, in a ladder-like fashion. The alternate β₁ conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure

Isotopically Site-Selected Dynamics of a Three-Stranded -Sheet Peptide Detected with Temperature-Jump IR-Spectroscopy

Infrared detected temperature jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded beta-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C=O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (XxxDPro amide) provided an alternate turn label as a control. Placing 13C=O labels on specific in-strand residues gave shifted modes that overlap the XxxDPro amide I’ modes. Their impact could be separated from the turn dynamics by a novel difference-transient analysis approach. FTIR spectra were modeled with DFTcomputations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with NMR structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, while the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, XxxDPro amides and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I’ and the second turn flipping from I’ to II’. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turnstabilized beta-sheet structure proceeds in a multi-step process

Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded β‑Sheet Peptides

A series of closely related peptide sequences that form triple-strand structures was designed with a variation of cross-strand aromatic interactions and spectroscopically studied as models for β-sheet formation and stabilities. Structures of the three-strand models were determined with NMR methods and temperature-dependent equilibrium studies performed using circular dichroism and Fourier transform infrared spectroscopies. Our equilibrium data show that the presence of a direct cross-strand aromatic contact in an otherwise folded peptide does not automatically result in an increased thermal stability and can even distort the structure. The effect on the conformational dynamics was studied with infrared-detected temperature-jump relaxation methods and revealed a high sensitivity to the presence and the location of the aromatic cross-links. Aromatic contacts in the three-stranded peptides slow down the dynamics in a site-specific manner, and the impact seems to be related to the distance from the turn. With a Xxx-^DPro linkage as a probe with some sensitivity for the turn, small differences were revealed in the relative relaxation of the sheet strands and turn regions. In addition, we analyzed the component hairpins, which showed less uniform dynamics as compared to the parent three-stranded β-sheet peptides

Vibrational circular dichroism sheds new light on the competitive effects of crowding and β-synuclein on the fibrillation process of α-synuclein

The effects of crowding, using the crowding agent Ficoll 70, and the presence of beta-synuclein on the fibrillation process of alpha-synuclein were studied by spectroscopic techniques, transmission electron microscopy and ThT-assays. This combined approach, where all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils while beta-synuclein leads to branched, short fibrils. The combi-nation of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the non-destructive technique of vibrational circular dichroism is extremely sensitive towards the length and the higher order morphology of the fibrils