Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

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.     

Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Redox-dependent changes in beta-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra.

The redox-dependent changes in secondary structure of cytochromes c from horse, cow, and dog hearts in water at 20 degrees C have been determined by amide I infrared spectroscopy. Second derivative amide I spectra were obtained by use of a procedure that includes a convenient method for the effective subtraction of the spectrum of water vapor in the system. The band at 1657 cm-1 representing the helix structure was unaffected by a change in redox state whereas changes in bands due to turns at 1680, 1672, and 1666 cm-1, unordered structure at 1650 cm-1, and beta-structures at 1632 and 1627 cm-1 occurred. About one-fourth of the beta-extended chain spectral region and one-fifth of the beta-turn region (involving a total of approximately 9-13 residues) were sensitive to the oxidation state of heme iron. No significant changes in the secondary structure of either the reduced or oxidized protein due to changes in ionic strength were detected. The localized structural rearrangements triggered by the changes in oxidation state of heme iron are consistent with differences in the binding of heme iron to a histidine imidazole nitrogen and a methionine sulfur atom from the beta-extended chain. The demonstrated ability to obtain highly reproducible second derivative amide I infrared spectra confirms the unique utility of such spectral measurements for localization of subtle changes in secondary structure within a protein, especially for changes among the multiple turns and beta-structures.     

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.     

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.     

FTIR-Spectroscopy of multistranded coiled coil proteins.

Coiled coils of different order were investigated using infrared (IR) spectroscopy. Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region.This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix. Here, we investigated coiled coils forming trimers, tetramers, and pentamers. These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion. We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra. However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils. The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A.     

Aggregation of chymotrypsinogen: portrait by infrared spectroscopy.

Changes in the secondary structure and aggregation of chymotrypsinogen were investigated by infrared difference spectroscopy in conjunction with temperature and pressure tuning IR spectroscopy; both the amide I' band and side chain bands were studied. A prominent component of the amide I' band in the difference spectrum obtained upon cooling a chymotrypsinogen solution, or increasing the hydrostatic pressure, was observed in the region between 1627 and 1622 cm-1. Under denaturing conditions a white gel was formed, which is attributed to irreversible self-association or aggregation. This process was accompanied by the appearance of two new amide I' bands in the infrared spectrum of the protein: a very strong band at 1618 cm-1 and a weak band at 1685 cm-1. These bands are assigned to peptide segments with anti-parallel aligned beta-strands.     

Infrared spectroscopic studies on gramicidin ion-channels: relation to the mechanisms of anesthesia

Fourier transform infrared spectroscopic studies are reported on gramicidin ion-channels in phospholipid bilayers and the effects on the spectra of the anesthetics and related compounds (methoxyflurane, halothane, chloroform, carbon tetrachloride, n-pentane and n-decane) have been determined. The addition of anesthetics containing the 'acidic hydrogen' caused unique changes particularly on the amide I bands at 1639 cm-1 and 1670 cm-1. The 1639 cm-1 band became more intense while the intensity near 1670 cm-1 decreased dramatically. These effects were not observed with carbon tetrachloride, n-pentane and n-decane. The 1670 cm-1 band is interpreted as arising from the carbonyls involved in the head-to-head hydrogen-bonded dimerization where the relationship between chains is analogous to that of the antiparallel beta-pleated sheet structure and the anesthetics with 'acidic hydrogens' are considered to disrupt the hydrogen-bonded dimerization by competitive hydrogen bonding to the carbonyls at the head-to-head junction. As the dimer-monomer equilibrium is the 'on-off' mechanism for gramicidin ion-channel conductance, the results are considered in terms of the mechanism of action of anesthetics and are taken to suggest, for certain anesthetics, a hydrogen-bonding role to protein ion-channel components.     

The quantitative analysis of uronic acid polymers by infrared spectroscopy.

I.r. absorption bands associated with the functional groups of carboxylic acid derivatives are useful for the analysis of alginates and pectins. The ester, amide, and uronate contents of pectins and the uronate content of alginates were determined, respectively, from the ester-carbonyl stretching band (1740 cm- minus 1), the amide I band (1650 cm- minus 1), and the carboxylate antisymmetric stretching band (1607 cm- minus 1) obtained from the spectra of solutions in D2O-phosphate buffer. The results are accurate to within plus or minus 2-4%, are self consistent, and agree well with the few reliable results that are available. The method should be applicable for the determination of carboxylic acid derivatives in other polysaccharides.     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.     

Estimation of beta-structure content of proteins by means of deconvolved FTIR spectra

Fourier self-deconvolution was applied to the infrared spectra of five globular proteins with a high beta-structure content and to the essentially alpha-helical protein hemoglobin. The featureless amide I' bands around 1650 cm-1 were thereby resolved into six to nine components, depending on the protein. Specific components were assigned to the beta-structure segments in each protein. The frequencies and the number of 'beta-bands' differ from one protein to another. The areas of the components were evaluated by means of a Gauss-Newton iteration procedure. It appears that the total area of the beta-bands, as a fraction of the total amide I' band area, reflects the relative beta-structure content of each protein studied.     

Infrared spectroscopy of a single cell--the human erythrocyte

Methods for obtaining the infrared spectrum of a single erythrocyte by infrared microscopy have been developed. The spectrum contains the amide I, II, and III bands characteristic of protein secondary structure near 1650, 1550, and 1300 cm-1, respectively. Bound carbon monoxide exhibits a readily measured band at 1951 cm-1 for 12C16O and 1907 cm-1 for 13C16O. Both amide and CO bands are similar to those found for purified hemoglobin A. Spectra can be obtained in H2O or D2O media under physiologically relevant conditions. Single cell infrared spectroscopy (SCIR) permits the qualitative and quantitative determination of differences among individual red cells. These results suggest many potential applications for SCIR for the measurements of properties of individual cells at the molecular level under physiologically relevant conditions.     

The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation.

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.     

An infrared spectroscopic study of the interactions of carbohydrates with dried proteins

Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of protein secondary structure from the laser Raman amide I spectrum

A new method for estimating protein secondary structure from the laser Raman spectrum has been developed whereby the amide I Raman band of a protein is analyzed directly as a linear combination of amide I bands of proteins whose secondary structures are known. For 14 proteins, analyzed by removing each one from the reference set and analyzing its structure in terms of the remaining proteins, the average correlation coefficients between the Raman and X-ray diffraction estimates of helix, beta-strand, turn, and undefined were 0.98, 0.98, 0.82 and 0.35, respectively. Significant correlations were also observed for distinctions between alpha-helix (0.98) and disordered helix (0.82), and between parallel (0.82) and antiparallel (0.97) beta-sheets. The average standard deviation of these Raman estimates from the X-ray values is less than 4%. In addition, a singular value analysis of 20 Raman amide I spectra indicates that there may be as many as nine significant independent pieces of information present in the amide I region.     

Temperature jump-induced secondary structural change of the membrane protein bacteriorhodopsin in the premelting temperature region: a nanosecond time-resolved Fourier transform infrared study

The secondary structural changes of the membrane protein, bacteriorhodopsin, are studied during the premelting reversible transition by using laser-induced temperature jump technique and nanosecond time-resolved Fourier transform infrared spectroscopy. The helical structural changes are triggered by using a 15 degrees C temperature jump induced from a preheated bacteriorhodopsin in D2O solution at a temperature of 72 degrees C. The structural transition from alphaII- to alphaI-helices is observed by following the change in the frequency of the amide I band from 1667 to 1651 cm-1 and the shift in the frequency of the amide II vibration from 1542 cm-1 to 1436 cm-1 upon H/D exchange. It is found that although the amide I band changes its frequency on a time scale of <100 ns, the H/D exchange shifts the frequency of the amide II band and causes a complex changes in the 1651-1600 cm-1 and 1530-1430 cm-1 frequency region on a longer time scale (>300 ns). Our result suggests that in this "premelting transition" temperature region of bacteriorhodopsin, an intrahelical conformation conversion of the alphaII to alphaI leads to the exposure of the hydrophobic region of the protein to the aqueous medium.     

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.     

Infrared microscopy for the study of biological cell monolayers. I. Spectral effects of acetone and formalin fixation

Infrared spectroscopy of biological cell monolayers grown on surfaces is a poorly developed field. This is unfortunate because these monolayers have potential as biological sensors. Here we have used infrared microscopy, in both transmission and transflection geometries, to study air-dried Vero cell monolayers. Using both methods allows one to distinguish sampling artefactual features from real sample spectral features. In transflection experiments, amide I/II absorption bands down-shift 9/4 cm(-1), respectively, relative to the corresponding bands in transmission experiments. In all other spectral regions no pronounced frequency differences in spectral bands in transmission and transflection experiments were observed. Transmission and transflection infrared microscopy were used to obtain infrared spectra for unfixed and acetone- or formalin-fixed Vero cell monolayers. Formalin-fixed monolayers display spectra that are very similar to that obtained using unfixed cells. However, acetone fixation leads to considerable spectral modifications. For unfixed and formalin-fixed monolayers, a distinct band is observed at 1740 cm(-1). This band is absent in spectra obtained using acetone-fixed monolayers. The 1740 cm(-1) band is associated with cellular ester lipids. In support of this hypothesis, two bands at 2925 and 2854 cm(-1) are also found to disappear upon acetone fixation. These bands are associated with C--H modes of the cellular lipids. Acetone fixation also leads to modification of protein amide I and II absorption bands. This may be expected as acetone causes coagulation of soluble cellular proteins. Other spectral changes associated with acetone or formalin fixation in the 1400-800 cm(-1) region are discussed. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 921-930, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.