Use of infrared spectroscopy to monitor protein structure and stability

One of the most versatile methods for monitoring the structure of proteins, either in solution or in the solid state, is Fourier transform infrared spectroscopy. Also known as mid-range infrared, which covers the frequency range from 4000 to 400 cm(-1), this wavelength region includes bands that arise from three conformationally sensitive vibrations within the peptide backbone (amide I, II and III). Of these vibrations, amide I is the most widely used and can provide information on secondary structure composition and structural stability. One of the advantages of infrared spectroscopy is that it can be used with proteins that are either in solution or in the solid state. The use of infrared to monitor protein structure and stability is summarized herein. In addition, specialized infrared methods are presented, such as techniques for the study of membrane proteins and oriented samples. In addition, there is a growing body of literature on the use of infrared to follow reaction kinetics and ligand binding in proteins, as well as a number of infrared studies on protein dynamics. Finally, the potential for using near-infrared spectroscopy to study protein structure is introduced.     

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.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Abstract Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium , on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.     

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy

The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm(-1). These results suggest that exposure to the ELF magnetic field has reversible effects on the N-H inplane bending and C-N stretching vibrations of peptide linkages, and changes the secondary structures of alpha-helix and beta-sheet in cell membrane proteins.     

Insight into the factors influencing the backbone dynamics of three homologous proteins,dendrotoxins I and K,and BPTI: FTIR and time-resolved fluorescence investigations

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties.     

A distinct utility of the amide III infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods

Fourier transform infrared spectroscopy is becoming an increasingly important method to study protein secondary structure. The amide I region of the protein infrared spectrum is the widely used region, whereas the amide III region has been comparatively neglected due to its low signal. Since there is no water interference in the amide III region and, more importantly, the different secondary structures of proteins have more resolved differences in their amide III spectra, it is quite promising to use the amide III region to determine protein secondary structure. In our current study, a partial least squares (PLS) method was used to predict protein secondary structures from the protein IR spectra. The IR spectra of aqueous solutions of 16 different proteins of known crystal structure have been recorded, and the amide I, the amide III, and the amide I combined with the amide III region of these proteins were used to set up the calibration set for the PLS algorithm. Our results correlate quite well with the data from X-ray studies, and the prediction from the amide III region is better than that from amide I or combined amide I and amide III regions.     

Laser Raman scattering of cobramine B, a basic protein from cobra venom

Cobramine B, a small basic protein from cobra venom, is selected as a model for studying the scattering intensity of tyrosyl ring vibrations in the Raman spectra of proteins. All three tyrosines in this protein appear to be “buried” in the interior of the molecule and probably involved in interactions which are similar to those of the three “buried” tyrosines in RNase A when it is dissolved in water. Spectral evidence is presented and discussed. The Raman spectra in the 300–1800 cm^?1 region of cobramine B in the solid and solution are compared quantitatively. Several differences exist between the two spectra and may be interpreted in terms of difference in conformation. In the amide I region, a strong single line was observed at 1672 cm^?1 both in the solid and solution spectra, suggesting that this protein may contain a large fraction of antiparallel-β structure. This is supported by the presence of a line at 1235 cm^?1 in the amide III region, which is also characteristic of β-structure. The resolved peaks at 1254 and 1270 cm^?1 indicate the coexistence of some hydrogen-bonded random-coil and some α-helix with the β-structure.     

Infrared techniques for quantifying protein structural stability

Biopharmaceutical and biotechnology companies and regulatory agencies require novel methods to determine the structural stabilities of proteins and the integrity of protein-protein, protein-ligand, and protein-membrane interactions that can be applied to a variety of sample states and environments. Infrared spectroscopy is a favorable method for a number of reasons: it is adequately sensitive to minimal sample amounts and is not limited by the molecular weight of the sample; yields spectra that are simple to evaluate; does not require protein modifications, a special supporting matrix, or internal standard; and is applicable to soluble and membrane proteins. In this paper, we investigate the application of infrared spectroscopy to the quantification of protein structural stability by measuring the extent of amide hydrogen/deuterium exchange in buffers containing D₂O for proteins in solution and interacting with ligands and lipid membranes. We report the thermodynamic stability of several protein preparations, including chick egg-white lysozyme, trypsin bound by benzamidine inhibitors, and cytochrome c interacting with lipid membranes of varying net-negative surface charge density. The results demonstrate that infrared spectroscopy can be used to compare protein stability as determined by amide hydrogen/deuterium exchange for a variety of cases.     

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.     

Structure of poly(ethylene glycol)-modified horseradish peroxidase in organic solvents: infrared amide I spectral changes upon protein dehydration are largely caused by protein structural changes and not by water removal per se

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.     

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.     

A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy

We have developed a holistic protein structure estimation technique using amide I band Raman spectroscopy. This technique combines the superposition of reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applicable to solution, suspension, and solid protein samples. A key component of this technique was the calculation of the reference spectra for ordered helix, unordered helix, and sheet, turns, and unordered structures from a series of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated samples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with previous experimental and theoretical results in the literature. We have demonstrated the use of these reference spectra for the estimation of secondary structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinations from X-ray crystallography is good.     

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Raman scattering of chymotrypsinogen A, ribonuclease, and ovalbumin in the aqueous solution and solid state

The Raman spectra of three globular proteins, beef pancreas chymotrypsinogen A, beef pancreas ribonuclease, and hen egg white ovalbumin have been obtained in the solid state and aqueous solution. X-ray diffraction and circular dichroism evidence have indicated that these proteins have a low α-helical content and a large fraction of the residues in the unordered and β-sheet conformation. The frequencies and intensities of the amide I and amide III lines are consistent with assignments based on the Raman spectra of polypeptides. The intense amide III lines observed in all the spectra would be expected for proteins with a low fraction of the residues in the α-helical conformation. Several spectra changes occur upon dissolution of the proteins in water and may be associated with further hydration of the proteins. The spectrum of thermally denatured chymotrypsinogen is presented. A 3 cm^–1 decrease in the frequency of the amide I line of the protein dissolved in D₂O upon heating was observed. This observation is consistent with a denaturation mechanism allowing only slight changes in the secondary structure but an increase in solvent penetration upon going from the native to the reversibly denatured state.     

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.     

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

Beware of proteins in DMSO

The effect on the secondary structure of representative alpha-helical, beta-sheet and disordered proteins by varying concentrations of dimethyl sulphoxide (DMSO) in 2H2O has been investigated by Fourier transform infrared spectroscopy. Significant perturbations of protein secondary structure are induced by DMSO and DMSO/2H2O mixtures. For highly structured proteins, such as myoglobin and concanavalin A, the infrared spectra point to a progressive destabilisation of the secondary structure until at moderate DMSO concentrations (around 0.33 mol fraction) intermolecular beta-sheet formation and aggregation are induced, as indicated by the appearance of a strong band at 1621 cm-1. This is a direct consequence of the disruption of intramolecular peptide group interactions by DMSO (partial unfolding). At higher DMSO concentrations (above 0.75 mol fraction), such aggregates are dissociated by disruption of the intermolecular C = O...2H-N deuterium bonds. The presence of a single amide I band at 1662 cm-1 corresponding to free amide C = O groups indicates that at high concentrations and in pure DMSO the proteins are completely unfolded, lacking any secondary structure. While low concentrations of DMSO showed no detectable effect upon the gross secondary structure of myoglobin and concanavalin A, the thermal stability of both proteins was markedly reduced. In alpha-casein, a highly unstructured protein, the situation is one of direct competition. The amide I maximum in 2H2O, at 1645 cm-1, is typical of unordered proteins with C = O groups deuterium-bonded predominantly to 2H2O. Addition of DMSO disrupts such interactions by competing with the peptide C = O group for the deuterium bond donor capacity of the 2H2O, and so progressively increases the amide I maximum until it stabilizes at 1663 cm-1, a position indicative of free C = O groups.