Evolution of quantitative methods in protein secondary structure determination via deep-ultraviolet resonance Raman spectroscopy

Deep-ultraviolet resonance Raman (DUVRR) spectra is sensitive to secondary structural motifs but, similar to circular dichroism (CD) and infrared spectroscopy, requires the application of multivariate and advanced statistical analysis methods to resolve the pure secondary structure Raman spectra (PSSRS) for determination of secondary structure composition. Secondary structure motifs are selectively enhanced by different excitation wavelengths, a characteristic that inspired the first methods for quantifying secondary structures by DUVRR. This review traces the evolution of multivariate methods and their application to secondary structure composition analyses of proteins by DUVRR spectroscopy from the first experiments using two-wavelengths, and culminating with recent studies utilizing time-resolved DUVRR measurements.     

Coherent two-dimensional infrared spectroscopy: quantitative analysis of protein secondary structure in solution

We present a method to quantitatively determine the secondary structure composition of globular proteins using coherent two-dimensional infrared (2DIR) spectroscopy of backbone amide I vibrations (1550-1720 cm(-1)). Sixteen proteins with known crystal structures were used to construct a library of 2DIR spectra, and the fraction of residues in α-helix, β-sheet, and unassigned conformations was determined by singular value decomposition (SVD) of the measured two-dimensional spectra. The method was benchmarked by removing each individual protein from the set and comparing the composition extracted from 2DIR against the composition determined from the crystal structures. To highlight the increased structural content extracted from 2DIR spectra a similar analysis was also carried out using conventional infrared absorption of the proteins in the library.     

Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: watching a protease eat a protein

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm(-1), suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm(-1) band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ～1594 cm(-1) and ～1406 cm(-1) increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.     

Thermally resolved synchrotron FT-IR microscopy of structural changes in bloodmeal-based thermoplastics

Synchrotron FT-IR micro-spectroscopy was used to determine thermally induced structural changes in thermoplastic protein produced from bloodmeal after mixing with sodium sulphite, sodium dodecyl sulphate, urea, tri-ethylene glycol and water. Changes in protein secondary structure at elevated temperature were assessed using second derivative peak height ratios in the amide III region (1,200–1,330 cm^?1) and compared with DSC and DMA results over the same temperature range. The results show an increase in ordered β-sheet structures with temperature at the expense of random coils, and that these β-sheets do not melt in the temperature range up to extrusion temperature of 120 °C. The implication of this is that during melt processing, β-sheet clusters may remain intact, similar to dispersed particulate fillers.     

2DIR spectroscopy of human amylin fibrils reflects stable β-sheet structure

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.     

Charge‐Induced Unzipping of Isolated Proteins to a Defined Secondary Structure

Here we present a combined experimental and theoretical study on the secondary structure of isolated proteins as a function of charge state. In infrared spectra of the proteins ubiquitin and cytochrome c, amide I (C=O stretch) and amide II (N–H bend) bands can be found at positions that are typical for condensed‐phase proteins. For high charge states a new band appears, substantially red‐shifted from the amide II band observed at lower charge states. The observations are interpreted in terms of Coulomb‐driven transitions in secondary structures from mostly helical to extended C₅‐type hydrogen‐bonded structures. Support for this interpretation comes from simple energy considerations as well as from quantum chemical calculations on model peptides. This transition in secondary structure is most likely universal for isolated proteins that occur in mass spectrometric experiments.     

Chiral sum frequency generation spectroscopy for characterizing protein secondary structures at interfaces

In situ and real-time characterization of protein secondary structures at interfaces is important in biological and bioengineering sciences, yet remains technically challenging. In this study, we used chiral sum frequency generation (SFG) spectroscopy to establish a set of vibrational optical markers for characterizing protein secondary structures at interfaces. We discovered that the N-H stretches along the peptide backbones of α-helices can be detected in chiral SFG spectra. We further observed that the chiral vibrational signatures of the N-H stretch together with the peptide amide I are unique to α-helix, β-sheet, and random coil at interfaces. Using these chiral vibrational signatures, we studied the aggregation of human islet amyloid polypeptide (hIAPP), which is implicated in type II diabetes. We observed in situ and in real time the misfolding of hIAPP from random coils to α-helices and then β-sheets upon interaction with a lipid-water interface. Our findings show that chiral SFG spectroscopy is a powerful tool to follow changes in protein conformations at interfaces and identify interfacial protein secondary structures that elude conventional techniques.     

Raman spectroscopy for forensic examination of β-ketophenethylamine “legal highs”: Reference and seized samples of cathinone derivatives

Raman spectra of a representative range of β-ketophenethylamine (β-KP), the rapidly growing family of cathinone-related “legal high” recreational drugs, have been recorded. These spectra showed characteristic changes that were associated with the pattern of substitution on the aromatic rings, for example, the compounds carrying substituents at the 4- position could be distinguished from 3,4-methylenedioxy “ecstasy” derivatives. They also showed small but detectable changes with differences in substitution on the ethylamine substituent. These features allowed the β-KPs present in seized casework samples to be identified. The seized samples typically contained only small amounts of bulking agents, which meant that the band intensities of these components within averaged data were very small. In contrast, grid sampling normally gave at least some spectra which had a higher than average proportion of the bulking agent(s), which allowed them to also be identified. This study therefore demonstrates that Raman spectroscopy can be used both to provide a rapid, non-destructive technique for identification of this class of drugs in seized samples and to detect minor constituents, giving a composition profile which can be used for drugs intelligence work.     

Structure and Composition of Insulin Fibril Surfaces Probed by TERS

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobson's model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002 , 99 , 9196 - 9201 ). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization.     

In situ Detection of Amide A Bands of Proteins in Water by Raman Ratio Spectrum

The amide A band of protein is sensitive to the hydrogen bands of amide groups of proteins. However, it is hard to distinguish the amide A band of aqueous protein in situ directly, since it overlaps with O-H stretching vibration of water. In this work, we presented a new analytical method of Raman ratio spectrum, which can extract the amide A band of proteins in water. To obtain the Raman ratio spectrum, the Raman spectrum of aqueous protein was divided by that of pure water. A mathematical simulation was employed to examine whether Raman ratio spectrum is effective. Two kinds of protein, lysozyme and α-chymotrypsin were employed. The amide A bands of them in water were extracted from Raman ratio spectra. Additionally, the process of thermal denaturation of lysozyme was detected from Raman ratio spectrum. These results demonstrated the Raman ratio spectra could be employed to study the amide A modes of proteins in water.     

三七总皂甙对牛血清白蛋白溶液构象的影响

应用衰减全反射傅立叶变换红外光谱结合荧光光谱和紫外光谱研究了中药三七 的有效成分三七总皂甙与牛血清白蛋白（BSA）的相互作用，采用对蛋白质红外光 谱酰氨Ⅰ带和酰氨Ⅲ带进行曲线拟合的方法，定量分析了不同浓度三七总皂甙对 BSA二级结构的影响，发现随着三七总皂甙浓度的增加，蛋白分子结构逐渐发生了 由螺旋向折叠的转化。a-螺旋结构减少了3%，β-折叠结构增加了约5%，其它二级 结构没有明显的变化，红外差谱和荧光光谱的结果为药物与蛋白质的作用引起牛血 清白蛋白溶液构象的变化提供了佐证，紫外光谱反映了单体皂甙与蛋白质的结合常 数的差异。     

Spectroscopic study of conformation changes of bovine serum albumin in aqueous environment

The secondary structural changes of protein aqueous solutions with and without calcium cations were investigated by attenuated total reflection-Fourier transform infrared (ATR-FTIR) technology.     

Vibrational spectral signatures of peptide secondary structures: N-methylation and side chain hydrogen bond in cyclosporin A

Molecular dynamics simulations are performed to explore important conformations of cyclosporin A, an immunosuppressive cyclic undecapeptide drug, in different media including gas-phase, chloroform, and acetonitrile. Density functional theory calculations are used to refine the low-lying conformers and to predict their infrared and vibrational circular dichroism spectra. Vibrational spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including β-turn (type II' or I), antiparallel β-sheet (flat or twisted), inverse γ-turn, N-methylated peptide bond, and side chain H-bond. New insights into the spectral signatures of secondary structures especially with N-methylation and side chain hydrogen bond are provided, which can be very useful for further peptide conformation analysis in general.     

Pre-processing of ultraviolet resonance Raman spectra

The application of UV excitation sources coupled with resonance Raman have the potential to offer information unavailable with the current inventory of commonly used structural techniques including X-ray, NMR and IR analysis. However, for ultraviolet resonance Raman (UVRR) spectroscopy to become a mainstream method for the determination of protein secondary structure content and monitoring protein dynamics, the application of multivariate data analysis methodologies must be made routine. Typically, the application of higher order data analysis methods requires robust pre-processing methods in order to standardize the data arrays. The application of such methods can be problematic in UVRR datasets due to spectral shifts arising from day-to-day fluctuations in the instrument response. Additionally, the non-linear increases in spectral resolution in wavenumbers (increasing spectral data points for the same spectral region) that results from increasing excitation wavelengths can make the alignment of multi-excitation datasets problematic. Last, a uniform and standardized methodology for the subtraction of the water band has also been a systematic issue for multivariate data analysis as the water band overlaps the amide I mode. Here we present a two-pronged preprocessing approach using correlation optimized warping (COW) to alleviate spectra-to-spectra and day-to-day alignment errors coupled with a method whereby the relative intensity of the water band is determined through a least-squares determination of the signal intensity between 1750 and 1900 cm(-1) to make complex multi-excitation datasets more homogeneous and usable with multivariate analysis methods.     

Structure and molecular conformation of tussah silk fibroin films: Effect of methanol

Structural changes of tussah (Antheraea pernyi) silk fibroin films treated with different water-methanol solutions at 20°C were studied as a function of methanol concentration and immersion time. X-ray diffraction measurements showed that the α-helix structure, typical of untreated tussah films, did not change for short immersion times (2 min), regardless of methanol concentration. However, crystallization to β-sheet structure was observed following immersion of tussah films for 30 min in methanol solutions ranging from 20 to 60% (v/v). IR spectra of tussah films untreated and methanol treated for 2 min exhibited strong absorption bands at 1265, 892, and 622 cm^?1, typical of the α-helix conformation. The intensity of the bands assigned to the β-sheet conformation (1245, 965, and 698 cm^?1) increased for the sample treated with 40% methanol for 30 min. Raman spectra of tussah films with α-helix molecular conformation exhibited strong bands at 1657 (amide I), 1263 (amide III), 1106, 908, 530, and 376 cm^?1. Following α → β conformational transition, amide I and III bands shifted to 1668, and to 1241, 1230 cm^?1, respectively. The band at 1106 cm^?1 disappeared and new bands appeared at 1095 and 1073 cm^?1, whereas the intensity of the bands at 530 and 376 cm^?1 decreased significantly. ©1995 John Wiley & Sons, Inc.     

UV resonance raman investigation of electronic transitions in alpha-helical and polyproline II-like conformations

UV resonance Raman (UVRR) excitation profiles and Raman depolarization ratios were measured for a 21-residue predominantly alanine peptide, AAAAA(AAARA) 3A (AP), excited between 194 and 218 nm. Excitation within the pi-->pi* electronic transitions of the amide group results in UVRR spectra dominated by amide vibrations. The Raman cross sections and excitation profiles provide information about the nature of the electronic transitions of the alpha-helix and polyproline II (PPII)-like peptide conformations. AP is known to be predominantly alpha-helical at low temperatures and to take on a PPII helix-like conformation at high temperatures. The PPII-like and alpha-helix conformations show distinctly different Raman excitation profiles. The PPII-like conformation cross sections are approximately twice those of the alpha-helix. This is due to hypochromism that results from excitonic interactions between the NV 1 transition of one amide group with higher energy electronic transitions of other amide groups, which decreases the alpha-helical NV 1 (pi-->pi*) oscillator strengths. Excitation profiles of the alpha-helix and PPII-like conformations indicate that the highest signal-to-noise Raman spectra of alpha-helix and PPII-like conformations are obtained at excitation wavelengths of 194 and 198 nm, respectively. We also see evidence of at least two electronic transitions underlying the Raman excitation profiles of both the alpha-helical and the PPII-like conformations. In addition to the well-known approximately 190 nm pi-->pi* transitions, the Raman excitation profiles and Raman depolarization ratio measurements show features between 205-207 nm, which in the alpha-helix likely results from the parallel excitonic component. The PPII-like helix appears to also undergo excitonic splitting of its pi-->pi* transition which leads to a 207 nm feature.     

Structural model of the amyloid fibril formed by beta(2)-microglobulin #21-31 fragment based on vibrational spectroscopy

A structural model of amyloid fibril formed by the #21-31 fragment of beta2-microglobulin is proposed from microscope IR measurements on specifically 13C-labeled peptide fibrils and Raman spectra of the dispersed fibril solution. The 13C-shifted amide frequency indicated the secondary structure of the labeled residues. The IR spectra have demonstrated that the region between F22 and V27 forms the core part with the extended beta-sheet structure. Raman spectra indicated the formation of a dimer with a disulfide bridge between C25 residues.     

Two-dimensional infrared spectroscopy of antiparallel beta-sheet secondary structure

We investigate the sensitivity of femtosecond Fourier transform two-dimensional infrared spectroscopy to protein secondary structure with a study of antiparallel beta-sheets. The results show that 2D IR spectroscopy is more sensitive to structural differences between proteins than traditional infrared spectroscopy, providing an observable that allows comparison to quantitative models of protein vibrational spectroscopy. 2D IR correlation spectra of the amide I region of poly-l-lysine, concanavalin A, ribonuclease A, and lysozyme show cross-peaks between the IR-active transitions that are characteristic of amide I couplings for polypeptides in antiparallel hydrogen-bonding registry. For poly-l-lysine, the 2D IR spectrum contains the eight-peak structure expected for two dominant vibrations of an extended, ordered antiparallel beta-sheet. In the proteins with antiparallel beta-sheets, interference effects between the diagonal and cross-peaks arising from the sheets, combined with diagonally elongated resonances from additional amide transitions, lead to a characteristic "Z"-shaped pattern for the amide I region in the 2D IR spectrum. We discuss in detail how the number of strands in the sheet, the local configurational disorder in the sheet, the delocalization of the vibrational excitation, and the angle between transition dipole moments affect the position, splitting, amplitude, and line shape of the cross-peaks and diagonal peaks.