Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm^?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm^?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm^?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm^?1 and an amide II band at 1548 cm^?1. The outer membrane had a broad amide I band at 1631–1657 cm^?1 and an amid II band at 1548 cm^?1 with a shoulder at 1520–1530 cm^?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm^?1, whereas the band at 1631 cm^?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.     

A holistic approach for protein secondary structure estimation from infrared spectra in H(2)O solutions

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.     

Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structures

Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra-structure correlations in Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.     

Conformation of poly-S-carboxyethyl-L-cysteine in aqueous solutions

Poly-S-carboxyethyl-L -cysteine, a higher side-chain homolog of poly-S-carboxymethyl-L -cysteine, has been prepared from poly-S-carbobenzoxyethyl-L -cysteine with hydrogen bromide in chloroform or acetic acid. The polymer is found to be in the β-conformation of an antiparallel arrangement of polypeptide chains in solid films, both in acid and salt forms, when examined by infrared spectra. Aqueous solutions of t he polymer have been investigated by measurements of rotatory dispersion and circular dichroism as well as by infrared spectra in D₂O. These properties show sharp changes around pH 5.5, as the pH of solution is varied. At higher ionization the polymer is randomly coiled, but at lower ionization it is in the β-conformation. Dependence of the rotatory properties upon polymer concentration as well as on ionic strength has been observed even at the lowest degree of ionization attained, and this has been attributed to the formation of intermolecular β-conformation in solutions. The β-structure is characterized by a negative circular dichroic band at 223 mμ and a positive dichroic band at a wavelength lower than 200 mμ, and furt her by a negative b_o value, ?140°. The pH-induced coil-β transition of the polymer is compared with that of poly-S-carboxymethl-L -cysteine.     

Determination of the secondary structure of proteins from the amide I band of the laser Raman spectrum

The amide I band in the laser Raman spectrum of proteins has been resolved into six components, each representing residues in a different type of secondary structure. These structure types are ordered or bihydrogen-bonded helix (believed to be located in the center of helical segments), disordered or monohydrogen-bonded helix (believed to be located at the ends of helical segments), antiparallel beta sheet, parallel beta sheet, reverse turn, and undefined. The Raman spectrum representing 100% of each type of residue conformation has been computed from the solvent-subtracted Raman spectra of ten proteins with known secondary structure, plus poly-l-lysine using a least-squares solution of the overdetermined system of equations. Linear combinations of these reference spectra were then fitted to the experimental amide I spectra of these and other proteins to estimate the fractions of residues in these conformations. Statistical tests suggest that the discrimination between bihydrogen-bonded helix and monohydrogen-bonded helix is significant as is the discrimination between parallel and antiparallel β-sheet. However, the discrimination between random structure and turns has not yet been accomplished by these studies. The absolute difference between X-ray and Raman estimates of structure for 17 protein samples is generally less than 6%. We conclude that detailed and reasonably accurate estimates of secondary structure can be derived from the amide I spectra of proteins.     

Computer analyses of characteristic infrared bands of globular proteins.

Infrared spectra of myoglobin, ribonuclease, lysozyme, α-chymotrypsin, α-lactalbumin, and β-lactoglobulin A were obtained in deuterium oxide solution in units of absorbance versus wavenumber from 1340 to 1750 cm^?1. The spectra were resolved into Gaussian components by means of an iterative computer program. Resolved characteristic absorption peaks for the two infrared active amide I′ components of antiparallel chain-pleated sheets (β-structure) were obtained. The characteristic amide I′ peaks of α-helical regions and apparently unordered regions overlap in D₂O solution. Absorptivity values for the resolved β-structure peak around 1630 cm^?1 were estimated on the basis of the known structure of ribonuclease, lysozyme, and β-chymotrypsin. The β-structure content of β-lactoglobulin was estimated to be ca. 48% of α-lactalbumin ca. 18%, and of α_s-casein close to zero. The results are in general agreement with conclusions drawn from circular dichroism and optical rotatory dispersion studies.     

Hierarchical band-target entropy minimization curve resolution and Pearson VII curve-fitting analysis of cellular protein infrared imaging spectra

A soft-modeling multivariate numerical approach that combines self-modeling curve resolution (SMCR) and mixed Lorentzian-Gaussian curve fitting was successfully implemented for the first time to elucidate spatially and spectroscopically resolved spectral information from infrared imaging data of oral mucosa cells. A novel variant form of the robust band-target entropy minimization (BTEM) SMCR technique, coined as hierarchical BTEM (hBTEM), was introduced to first cluster similar cellular infrared spectra using the unsupervised hierarchical leader-follower cluster analysis (LFCA) and subsequently apply BTEM to clustered subsets of data to reconstruct three protein secondary structure (PSS) pure component spectra—α-helix, β-sheet, and ambiguous structures—that associate with spatially differentiated regions of the cell infrared image. The Pearson VII curve-fitting procedure, which approximates a mixed Lorentzian-Gaussian model for spectral band shape, was used to optimally curve fit the resolved amide I and II bands of various hBTEM reconstructed PSS pure component spectra. The optimized Pearson VII band-shape parameters and peak center positions serve as means to characterize amide bands of PSS spectra found in various cell locations and for approximating their actual amide I/II intensity ratios. The new hBTEM methodology can also be potentially applied to vibrational spectroscopic datasets with dynamic or spatial variations arising from chemical reactions, physical perturbations, pathological states, and the like.     

Relationship between digestibility and secondary structure of raw and thermally treated legume proteins: a Fourier transform infrared (FT-IR) spectroscopic study

The secondary structure of proteins in legumes, cereals, milk products and chicken meat was studied by diffuse reflectance infrared spectroscopy in the region of the amide I band. Major secondary structure components ( β-sheets, random coil, α-helix, turns), together with the low- and high-frequency side contributions, were resolved and related to the in vitro digestibility behaviour of the different foods. A strong inverse correlation between the relative spectral weights of the β-sheet structures and in vitro protein digestibility values was measured. Structural modifications in legume proteins induced by autoclaving were monitored by the changes in the amide I spectra. The results indicate that the β-sheet structures of raw legume proteins and the intermolecular β-sheet aggregates, arising upon heating, are primary factors in adversely affecting the digestibility.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide II infrared bands. Comparison between classical and partial least-squares methods

A method for estimating protein secondary structure from infrared spectra has been developed. The infrared spectra of H2O solutions of 13 proteins of known crystal structure have been recorded and corrected for the spectral contribution of water in the amide I and II region by using the algorithm of Dousseau et al. [Dousseau, F., Therrien, M., & Pézolet, M. (1989) Appl. Spectrosc. 43, 538-542]. This calibration set of proteins has been analyzed by using either a classical least-squares (CLS) method or the partial least-squares (PLS) method. The pure-structure spectra calculated by the classical least-squares method are in good agreement with spectra of poly(L-lysine) in the alpha-helix, beta-sheet, and undefined conformations. The results show that the best agreement between the secondary structure determined by X-ray crystallography and that predicted by infrared spectroscopy is obtained when both the amide I and II bands are used to generate the calibration set, when the PLS method is used, and when it is assumed that the secondary structure of proteins is composed of only four types of structure: ordered and disordered alpha-helices, beta-sheet, and undefined conformation. Attempts to include turns in the secondary structure estimation have led to a loss of accuracy. The standard deviation of the difference between X-ray and infrared secondary structure estimates with this method is 4.8% for the alpha-helix, 3.7% for the beta-sheet, and 5.1% for the undefined structure, whereas the regression coefficients are 0.95, 0.96, and 0.56, respectively. The spectra of the calibration proteins were also recorded in 2H2O solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infrared spectra and resonance interaction of amide-I vibration of the antiparallel-chain pleated sheet.

Theoretical treatment of resonance interaction of amide-I vibration has been done in a dipole–dipole approximation on the basis of perturbation theory. A single infinite antiparallel-chain pleated sheet as well as different kinds of its finite fragments have been considered. A good agreement has been obtained between calculated spectral parameters of amide I of the infinite sheet and observed ones in infrared and Raman spectra of synthetic polypeptides and fibrous proteins. A theoretical dependence of the resonance frequency shift of the main component and frequency splitting of two components active in the infrared spectra on the number of polypeptide chains in the finite sheet has been found.     

The optimization of protein secondary structure determination with infrared and circular dichroism spectra.

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.     

Infrared spectra and resonance interaction of amide-I vibration of the paraellel-chain pleated sheets.

Resonance vibrational interactions of amide I for the parallel-chain pleated-sheet structure have been treated on the basis of the perturbation theory in a dipole–dipole approximation. The infinite sheet and finite fragments of different types have been considered. The possibility of experimental observation by infrared spectra of parallel-chain pleated-sheet fragments in globular proteins is discussed.     

Fourier transform infrared studies of ribonuclease in H2O and 2H2O solutions

Fourier transform infrared transmission spectra have been obtained of the enzyme ribonuclease in both H₂O and ²H₂O. The resolution of the spectra have been enhanced by Fourier self-deconvolution procedures. The infrared spectrum of ribonuclease changes during exchange of the enzyme's amide hydrogens for deuterium and the exchange has been followed in the amide I and amide II spectral regions. The amide I band shifts towards lower wavenumbers during both the fast and slow phases of hydrogen exchange and the interpretation of these shifts has aided the band assignments. In particular these studies have allowed an assignment to be made for the high frequency component of the β-strand absorption that differs from that proposed previously. This paper represents the first example of the use of deconvoluted Fourier transform infrared spectra in conjunction with hydrogen-deuterium exchange in order to aid in the assignment of a proteins's infrared bands.     

Automatic amide I frequency selection for rapid quantification of protein secondary structure from Fourier transform infrared spectra of proteins

Here we report the development of a new neural network based approach for rapid quantification of protein secondary structure from Fourier transform infrared (FTIR) spectra of proteins. A technique for efficiently reducing the amount of spectral data by almost 90% is suggested to facilitate faster neural network analysis. Additionally, an automatic procedure is introduced for selecting only those regions within the amide I band of protein FTIR spectra, which can be best related to secondary structure contents by subsequent neural network analysis. Based on a given reference set of FTIR spectra from proteins with known secondary structure, a subset of merely 29 out of 101 amide I absorbance values could be identified, which lead to an improved prediction accuracy. The average prediction accuracy achieved for helix, sheet, turn, bend, and other is 4.96% which is better than that achieved by alternative methods that have been previously reported indicating the significant potential of this approach. Our suggested automatic amide I frequency selection procedure may be easily extended to identify promising regions from spectral data recorded by other spectroscopic techniques, like for example circular dichroism spectroscopy.     

A representation learning approach for recovering scatter-corrected spectra from Fourier-transform infrared spectra of tissue samples

Infrared spectra obtained from cell or tissue specimen have commonly been observed to involve a significant degree of scattering effects, often Mie scattering, which probably overshadows biochemically relevant spectral information by a nonlinear, nonadditive spectral component in Fourier transform infrared (FTIR) spectroscopic measurements. Correspondingly, many successful machine learning approaches for FTIR spectra have relied on preprocessing procedures that computationally remove the scattering components from an infrared spectrum. We propose an approach to approximate this complex preprocessing function using deep neural networks. As we demonstrate, the resulting model is not just several orders of magnitudes faster, which is important for real-time clinical applications, but also generalizes strongly across different tissue types. Using Bayesian machine learning approaches, our approach unveils model uncertainty that coincides with a band shift in the amide I region that occurs when scattering is removed computationally based on an established physical model. Furthermore, our proposed method overcomes the trade-off between computation time and the corrected spectrum being biased towards an artificial reference spectrum.