Structure of Condensed Phase Peptides: Insights from Vibrational Circular Dichroism and Raman Optical Activity Techniques.

Peptides and proteins are naturally chiral molecular systems so that sensing their structure and conformation with chirality-based spectral methods is an obvious and long-used diagnostic application. Extending chiroptical techniques to measurement of vibrational transitions, in the form of vibrational circular dichroism (VCD) and Raman optical activity (ROA), expands the number and types of excitations available that might provide structural insight and can provide an alternate and, in some cases, a more distinctive conformational probe. Since the dominant repeating structural element in peptides is the locally achiral amide group, VCD senses the polymeric structure through amide coupling, which is directly dependent on secondary structure. Determination of the type and relative contribution of these structural components through empirical correlation with spectral character has been the main application of VCD for peptides and proteins, although this is now reinforced by extensive theoretical modeling. Monitoring structural and conformational change induced by environmental perturbations provides another important application. More recently, VCD has been used to detect morphological variations in fibril states of aggregated peptides and proteins. ROA has parallel secondary structural sensitivities, with more applications for proteins than peptides, and has more sensitivity to local configuration and side chains. This review covers the range of peptide studies done with VCD and extends them to compare with example protein and ROA applications.

Monitoring peptide tyrosine nitration by spectroscopic methods.

Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.

Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature-Jump IR Spectroscopy and Isotope Labeling.

Site-specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded ß-sheet peptide and by use of selected 13 C isotope substitutions at multiple positions in the peptide backbone. Single-strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross-strand double- and triple-labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local-mode coupling. Temperature-jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site-specific relaxation rates were altered upon the introduction of additional cross-strand isotopes. Likewise, the rates for the global ß-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.

Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and ß-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of ß-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which 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 ß-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

Theory of Molecular Vibrational Zeeman Effects as Measured with Circular Dichroism.

We present a general theory that enables the first nonempirical computation of molecular vibrational Zeeman effects as are detectable with magnetic vibrational circular dichroism spectroscopy (MVCD). In this method, the second derivatives of the molecular magnetic moment appear to be essential to determine the observable MVCD intensities. Using a quasiharmonic approximation, computations based on our method allowed a band-to-band comparison of simulated to measured spectra. Given this new possibility of its reliable interpretation, MVCD spectroscopy may develop as a useful tool to yield detailed information on molecular vibrational states and structure, including achiral systems.

Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded ß-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 ß-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 ß-sheet peptides.

Mini review: Instrumentation for vibrational circular dichroism spectroscopy, still a role for dispersive instruments.

Vibrational circular dichroism (VCD) has become a standard method for determination of absolute stereochemistry, particularly now that reliable commercial instrumentation has become available. These instruments use a now well-documented Fourier transform infrared-based approach to measure VCD that has virtually displaced initial dispersive infrared-based designs. Nonetheless, many papers have appeared reporting dispersive VCD data, especially for biopolymers. Instrumentation designed with these original methods, particularly after more recent updates optimizing performance in selected spectral regions, has been shown still to have advantages for specific applications. This article presents a mini-review of dispersive VCD instrument designs and includes sample spectra obtained for various biopolymer (particularly peptide) samples. Complementary reviews of Fourier transform-VCD designs are broadly available.

Instrumentation for Vibrational Circular Dichroism Spectroscopy: Method Comparison and Newer Developments.

Vibrational circular dichroism (VCD) is a widely used standard method for determination of absolute stereochemistry, and somewhat less so for biomolecule characterization and following dynamic processes. Over the last few decades, different VCD instrument designs have developed for various purposes, and reliable commercial instrumentation is now available. This review will briefly survey historical and currently used instrument designs and describe some aspects of more recently reported developments. An important factor in applying VCD to conformational studies is theoretical modeling of spectra for various structures, techniques for which are briefly surveyed.

Reduced and mutant lysozyme refolding with lipid vesicles. Model study of disulfide impact on equilibria and dynamics.

The recovery of secondary structure in disordered, disulfide-reduced hen egg white lysozyme (HEWL) upon interaction with lipid vesicles was studied using circular dichroism (CD), fluorescence and infrared (IR) spectroscopic techniques. Lipid vesicles having negative head groups, such as DMPG, interact with reduced HEWL to induce formation of more helical structure than in native HEWL, but no stable tertiary structure was evident. Changes in tertiary structure, as evidenced by local environment of the tryptophan residues, were monitored by fluorescence. Spectra for oxidized HEWL, reduced HEWL and mutants with no or just one disulfide bond developed variable degrees of increased helicity when added to negatively charged lipid vesicles, mostly depending on packing of tails. When mixed with zwitterionic lipid vesicles, reduced HEWL developed ß-sheet structure with no change in helicity, indicating an altered interaction mechanism. Stopped flow CD and fluorescence dynamics, were fit to multi-exponential forms, consistent with refolding to metastable intermediates of increasing helicity for HEWL interacting with lipid vesicles. Formation of an intermediate after rapid interaction of the lipid vesicles and the protein is supported by the correlation of faster steps in CD and fluorescence kinetics, and largely appears driven by electrostatic interaction. In subsequent slower steps, the partially refolded intermediate further alters structure, gaining helicity and modifying tryptophan packing, as driven by hydrophobic interactions.

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif.

ß-Barrelmembrane proteins (ßMPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, ßMPs have great potential as nanosensors for single-molecule detection. However, natural ßMPs currently employed have inflexible biophysical properties and are limited in their pore geometry, hindering their applications in sensing molecules of different sizes and properties. Computational engineering has the promise to generate ßMPs with desired properties. Here we report a method for engineering novel ßMPs based on the discovery of sequence motifs that predominantly interact with the cell membrane and appear in more than 75% of transmembrane strands. By replacing ß1-ß6 strands of the protein OmpF that lack these motifs with ß1-ß6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel ßMP called OmpGF. OmpGF is predicted to form a monomer with a stable transmembrane region. Experimental validations showed that OmpGF could refold in vitro with a predominant ß-sheet structure, as confirmed by circular dichroism. Evidence of OmpGF membrane insertion was provided by intrinsic tryptophan fluorescence spectroscopy, and its pore-forming property was determined by a dye-leakage assay. Furthermore, single-channel conductance measurements confirmed that OmpGF function as a monomer and exhibits increased conductance than OmpG and OmpF. These results demonstrated that a novel and functional ßMP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation.

Structural Rearrangement from Oligomer to Fibril Detected with FRET in a Designed Amphiphilic Peptide.

ß-Sheet conformation is promoted in peptides with amphiphilic design, and stable ß-turn formation is favored with the unnatural amino acid d-Pro followed by a flexible residue such as Gly. A 19-residue peptide (B3) was synthesized with alternating hydrophobic and hydrophilic residues connected by symmetrical d-Pro-Gly and Gly-d-Pro turns. B3 forms an oligomeric aggregate, rich in ß-sheet conformation, that reversibly transforms into an unordered structure on heating, as evidenced by its temperature-dependent IR spectra. When a dansyl moiety was added to the N terminus of B3, the resulting peptide (B3D) can convert into a fibrillar structure after higher temperature incubation, as detected spectroscopically as well as by TEM. The fibrillization process involves an initial unfolding step monitored by the quenching of dansyl emission; in contrast, subsequent enhanced thioflavin T emission is seen on its binding to the fibril. A possible mechanism is proposed: B3D forms a low-temperature oligomer, which is at least partially unfolded by heat and subsequently reassembles more slowly as a fibril.

Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra.

Site-Specific Dynamics of ß-Sheet Peptides with (D) Pro-Gly Turns Probed by Laser-Excited Temperature-Jump Infrared Spectroscopy.

Turn residues and side-chain interactions play an important role for the folding of ß-sheets. We investigated the conformational dynamics of a three-stranded ß-sheet peptide ((D) P(D) P) and a two-stranded ß-hairpin (WVYY-(D) P) by time-resolved temperature-jump (T-jump) infrared spectroscopy. Both peptide sequences contain (D) Pro-Gly residues that favor a tight ß-turn. The three-stranded ß-sheet (Ac-VFITS(D) PGKTYTEV(D) PGOKILQ-NH2 ) is stabilized by the turn sequences, whereas the ß-hairpin (SWTVE(D) PGKYTYK-NH2 ) folding is assisted by both the turn sequence and hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, (D) P(D) P being faster than WVYY-(D) P. The Xxx-(D) Pro tertiary amide provides a detectable IR band, allowing us to probe the dynamics site-specifically. The relative importance of the turn versus the intrastrand stability in ß-sheet formation is discussed.

Role of Side Chains in ß-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 ß2-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 ß-branched aliphatic residues, Val and Ile, that could potentially pack with Glu side chains, these oligopeptides do show evidence of ß2-stacking. By contrast, for Leu, with longer side chains, only ß1-stacking is seen while with even larger Phe side chains, either ß-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 ß1- and ß2-structures, that overall suggests two different packing modes for the hydrophobic side chains depending on size and type.

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 ß-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.

Equilibrium and dynamic spectroscopic studies of the interaction of monomeric ß-lactoglobulin with lipid vesicles at low pH.

ß-Lactoglobulin (ßLG) 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 ßLG is dimeric. In this study, a ß-sheet to α-helix transformation was also observed for monomeric ßLG 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-sn-glycero-3-phosphocholine (DSPC), at ~50 °C and pH 2.6, the ßLG 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 ßLG 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 ßLG 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 ßLG with zwitterionic lipids different from that seen previously for the dimeric form.

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.

Increased accuracy of vibrational circular dichroism calculations for isotopically labeled helical peptides.

Vibrational circular dichroism (VCD) spectra have been computed with qualitatively correct sign patterns for α-helical peptides using various methods, ranging from empirical models to ab initio quantum mechanical computations. However, some details, such as deuteration effects and isotope substitution shifts and sign patterns for the resultant amide I' band shape, have remained a predictive challenge. Fully optimized computations for a 25-residue Ala-rich peptide, including implicit solvent corrections and explicit side chains that experimentally stabilize these model helical peptides in water, have been carried out using density functional theory (DFT). These fully minimized structures show minor changes in the (ϕ,ψ) torsions at the termini and yield an extra negative band to the low energy side of the characteristic amide I' couplet VCD, in agreement with experiments. Additionally, these calculations give the right sign and relative intensity patterns, as compared to experimental results, for several 13C=O substituted variants. The differences from previously reported computations that used ideal helical structures and vacuum conditions imply that inclusion of distorted termini and solvent effects can have an impact on the final detailed spectral patterns. Inclusion of side chains in these calculations had very little effect on the computed amide I' IR and VCD. Tests of constrained geometries, varying dielectric, and different functionals indicate that each can affect the band shapes, particularly for the 12C=O components, but these aspects do not fully explain the difference from previous spectral simulations. Inclusion of long-range amide coupling, as obtained from DFT computation of the full structure, or transfer of parameters from a somewhat longer peptide model, rather than shorter model, seems to be more important for the final detailed band shape under isotopic substitution. However, these corrections can also induce other changes, suggesting that previously reported, limited calculations may have been qualitatively useful due to a balance of errors. This may also explain the success of simple empirical IR models.

Insight into the packing pattern of ß2 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 ß2-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 (13)C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and ß2 aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional ß1-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 ß2 structure is based on stacked, twisted, antiparallel ß-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 ß1 conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure.

Role of different ß-turns in ß-hairpin conformation and stability studied by optical spectroscopy.

Model ß-hairpin peptides based on variations in the turn sequence of Cochran's tryptophan zipper peptide, SWTWENGKWTWK, were studied using electronic circular dichroism (ECD), fluorescence, and infrared (IR) spectroscopies. The trpzip2 Asn-Gly turn sequence was substituted with Thr-Gly, Aib-Gly, (D)Pro-Gly, and Gly-Asn (trpzip1) to study the impact of turn stability on ß-hairpin formation. Stability and conformational changes of these hairpins were monitored by thermodynamic analyses of the temperature variation of both FTIR (amide I') and ECD spectral intensities. These changes were fit to a two-state model which yielded different T(m) values, representing the folding/unfolding process, for hairpins with different ß-turns. Different ß-turns show systematic contributions to hairpin structure formation, and their inclusion in hairpin design can modify the folding pathways. Aib-Gly or (D)Pro-Gly sequences stabilize the turn resulting in residual Trp-Trp interaction at high temperatures, but at the same time the ß-structure (cross strand H-bonds) can become less stable due to constraints of the turn, as seen for (D)Pro-Gly. The structure of the Aib-Gly turn containing hairpin was determined by NMR and was shown to be like trpzip2 (Asn-Gly turn) as regards turn and strand geometries, but to differ from trpzip1 (Gly-Asn turn). The Munoz and Eaton statistical mechanically derived multistate model, tested as an alternate point of view, represented contributions from H-bonds and hydrophobic interactions as well as conformational change as interdependent. Use of different spectral methods that vary in dependence on these physical interactions along with the structural variations provided insight to the complex folding pathways of these small, well-folded peptides.