Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm-1 in infrared spectra of dried protein-sugar samples was examined. For samples in which a high level of native protein structure was retained in the dried solid, the apparent moisture content predicted by the carboxylate band area was greater than the actual moisture content, indicating that protection results from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural protection conferred by sucrose and trehalose apparent in second derivative, amide I infrared spectra, correlates with the extent of hydrogen bonding between sugar and protein. The failure of dextran to inhibit dehydration-induced lysozyme unfolding is shown to result from the inability of the polymer to hydrogen bond adequately to the protein. Therefore, formation of an amorphous phase alone is not sufficient to maintain protein structure during dehydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryoprotectant. However, the addition of polyethylene glycol, which is known to protect proteins during freezing, but not drying, to glucose protected lysozyme structure during lyophilization. Together, these results show that hydrogen bonding between carbohydrate and protein is necessary to prevent dehydration-induced protein damage. However, hydrogen bonding alone is not sufficient to protect proteins during lyophilization in the absence of adequate freezing protection.     

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane

Infrared spectroscopy was used to investigate the changes in bands assigned to phospholipids and proteins in dehydrated and rehydrated sarcoplasmic reticulum. The changes in CH₂ and CH₃ stretching bands, amide bands, and phosphate stretching bands are similar to shifts in frequency seen for those bands in phospholipid and protein preparations during thermotropic phase transitions and hydration. IR studies on dry trehalose-sarcoplasmic reticulum mixtures show similar results; with increasing trehalose concentration in the dry mixtures, amide and phosphate bands shift to frequencies characteristic of hydrated samples. Changes in bands assigned to OH deformations in the trehalose suggest that the interaction between the carbohydrate and membrane is by means of hydrogen bonding between these OH groups and membrane components.     

Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRAS) of fibrinogen adsorbed on metal and metal oxide surfaces

We have investigated the possibilities of using Infrared Reflection Absorption Spectroscopy in the study of the interaction of proteins with metal surfaces. Structural information can be obtained since the infrared radiation at the metal surface interacts only with dipole transition moments perpendicular to the metal surface. Fibrinogen spontaneously adsorbed from solution onto gold, titanium and aluminum was used as model systems. The infrared studies were carried out on dried protein films. The amide I bands of fibrinogen adsorbed on the metal surfaces shift towards higher frequencies (ca. 20 cm-1) relative to the same band in buffer solution. The magnitude of these shifts indicates that conformational change of the protein occurs upon adsorption on metal surfaces. The change in conformation of the fibrinogen also can partly be due to one week of drying at room temperature. The amide I and amide II bands show a slightly different behaviour in terms of frequency and intensity for each metal-protein system studied. The side chains appeared to be more substrate sensitive than the peptide group. Orientational effects were observed for a number of side-chain related groups.     

Low-temperature IR spectroscopy reveals four stages of water loss during lyophilization of hen egg white lysozyme

Water loss during lyophilization of a 49.4 mg/mL solution of lysozyme in D₂O was studied with ir spectroscopy using a low-temperature, single reflection, horizontal, attenuated, total reflectance accessory. Four regions of water loss were identified and assignable to different forms of bound water. The amide I band begins to shift to higher frequency while the amide II concurrently shifts to lower frequency and broadens after the first stage of water loss (sublimation) at ?10°C. Additionally, the carboxylate band (at 1584 cm^?1) shifts slightly to lower frequency. A second stage at 17°C is characterized by continued shifts in the carboxylate and amide II bands to low frequency, further broadening in the amide II and greater shift to high frequency in the amide I (ascribed to the removal of periphery water around the protein). At the third stage of water loss, the carboxylate band decreases substantially in relative absorbance (consistent with the removal of water from the carbonyl backbone). In the fourth and last stage, the carboxylate band nearly disappears and water loss is very slow. Based upon a final level of hydration of 0.037 h, the last stage corresponds to 25% completion of the removal of water associated with ionizable side chains. From start to finish, the amide I shifts 9 cm^?1 to higher frequency. © 1994 John Wiley & Sons, Inc.     

Interaction of carbohydrates with dry dipalmitoylphosphatidylcholine

Interactions of six carbohydrates (trehalose, sucrose, glucose, raffinose, inositol, and glycerol) with dry dipalmitoylphosphatidylcholine (DPPC) were studied using differential scanning calorimetry (DSC) and infrared spectroscopy (ir) in order to elucidate the mechanism by which some of these carbohydrates preserve structural and functional integrity of dry membranes. Results with DSC showed that trehalose depressed the main transition temperature (Tmid) of dry DPPC below that of fully hydrated DPPC, and raised the enthalpy of that transition more than did addition of water. Results obtained with ir spectroscopy suggested a potential mechanism for this interaction. In the presence of most of the carbohydrates the ir spectrum for DPPC showed changes similar to those seen when water was added to dry DPPC, and the asymmetric P = O stretching band was diminished in intensity. The degree to which the carbohydrates tested affected the integrated intensity of this band and the Tmid was correlated with the ability of those carbohydrates to preserve dry membranes. Also, bands assigned to -OH deformations in the trehalose and other carbohydrates were depressed in the presence of DPPC. Based on these observations, it is suggested that the mechanism of interaction between the carbohydrate and lipid involves hydrogen bonding between -OH groups on the carbohydrate and the phosphate head group of the phospholipid. The only exceptions to this pattern are glycerol, which depresses Tmid of dry DPPC, and myo-inositol, which has no effect on Tmid or the ir spectrum of DPPC; neither carbohydrate can preserve dry membranes. It is suggested, based on ir spectroscopy and previous results with monolayer preparations, that glycerol interacts with phospholipids by a mechanism different from that shown by the other carbohydrates.     

Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.     

Polarized infrared attenuated total reflectance spectroscopy of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca(2+)-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl2, 1-10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing congruent to 7.5 micrograms protein/cm2, and decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH2 vibrations (2923 cm-1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm-1) of the Ca(2+)-ATPase in the Ca2-E1 state and in the EGTA and vanadate stabilized E2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH2 vibrations was not affected by changes in the concentration of KCl (25-100 mM) or Ca2+ (approximately equal to 10(-8)-10(-4) M) and by the addition of vanadate (1 mM) or Pi (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm-1) or CO stretching band (1046 cm-1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of approximately 70 degrees between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca(2+)-ATPase.     

IR and Raman spectroscopic studies of the interaction of trehalose with hen egg white lysozyme

Infrared and Fourier transform Raman spectra are reported for dried mixtures of trehalose and lysozyme. The Raman spectra show effects on the protein amide I band and some sugar bands that are not present when the components are dried separately. Comparison of ir spectra with those published previously show significant differences. It is concluded that these arise because of differences in the extent of drying of the moisture, and that, contrary to some claims, vibrational spectroscopy does not so far show any clear evidence of specific trehalose/protein interactions and that results may be interpreted in terms of entrapment of water within the mixture. © 1994 John Wiley & Sons, Inc.     

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.     

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.     

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.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

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.     

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.     

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.     

Fourier transform infrared study of proteins with parallel beta-chains

Deconvolved and second derivative Fourier transform infrared spectra of the proteins flavodoxin and triosephosphate isomerase have been obtained in the 1600 to 1700 cm-1 (amide I) region. To our knowledge these results provide the first experimental infrared data on proteins with parallel beta-chains. Characteristic absorption bands for the parallel beta-segments are observed at 1626-1639 cm-1 (strong) and close to 1675 cm-1 (weak). Previous theoretical studies based on hypothetical models with large, regular beta-sheets had suggested bands close to 1650 and 1666 cm-1. Our new assignments were confirmed by band area measurements, which yield conformational information in good agreement with results from X-ray diffraction data. The spectra were compared with corresponding spectra of concanavalin A and carboxypeptidase A. The first contains only antiparallel beta-segments, the second "mixed" beta-segments, with some strands lying antiparallel and others parallel. None of the observed amide I band frequencies assigned to parallel beta-chains occurs in the 1650 cm-1 region associated with helical segments.     

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.     

Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy.

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.