Fourier-transform infrared spectroscopy study of the pressure-induced changes in the structure of the bovine alpha-lactalbumin: the stabilizing role of the calcium ion.

The Fourier-transform infrared spectroscopy (FTIR) technique with a diamond anvil cell has been applied for examination of the pressure-induced changes occurring in the secondary structure of the alpha-lactalbumin. This is the first high-pressure FTIR study of a calcium-binding protein which simultaneously takes into account spectral changes in both the calcium-ion-binding carboxyl groups' band and the amide I/I' vibrational band. Spectral behavior of three kinds of the protein: the undeuterated holoform, the fully deuterated holoform, and the undeuterated apoform was compared in the pressure range from 0.1 MPa up to 740 MPa. We found that the binding of calcium remarkably stabilizes the alpha-lactalbumin against pressure as it is followed approximately by a 200-MPa increase of the value of pressure at which denaturation occurs. A quantitative analysis of the band of antisymmetrical stretching vibrations of the calcium-binding carboxyl groups revealed that the pressure-induced changes in the calcium-binding loop occur in two stages. Binding of the calcium ion seemingly increases the pressure-stability of the calcium-binding loop to a higher degree than the pressure-stability of the secondary structure of the alpha-lactalbumin. We have also discussed in detail the complex pressure-enhanced H/D exchange in the alpha-lactalbumin. Finally, we have proposed a new assignment of major peaks in the helical region of the amide I/I' spectral band of the partially deuterated alpha-lactalbumin.     

IR spectroscopy of isotope-labeled helical peptides: probing the effect of N-acetylation on helix stability

The effect of N-acetylation on the conformation of alanine-rich helical peptides is examined using isotope-edited Fourier transform infrared (FTIR) spectroscopy. A series of peptides with sequence AA(AAKAA)(3)AAY has been prepared; each peptide incorporates four (13)C-labeled alanines. These peptides have two amide I' bands in their FTIR spectra: one corresponding to the (12)C amino acids, and one assigned to the (13)C amino acids. The intensity and frequency of the (13)C amide I' band varies systematically with the position of the labels in the sequence and the presence or absence of an N-acetyl capping group. The intensity of the (13)C amide I' band correlates with helix stability at the labeled residues as predicted by thermodynamic models of the helix-coil transition. These results suggest that FTIR spectroscopy combined with specific isotope labeling can be used to dissect the conformation of helical peptides at the residue level.     

Induction of secondary structure in a COOH-terminal peptide of histone H1 by interaction with the DNA: an infrared spectroscopy study.

We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1(0) (residues 99-121) and is adjacent to the central globular domain of the protein. In aqueous (D(2)O) solution the amide I' is dominated by component bands at 1643 cm(-1) and 1662 cm(-1), which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm(-1)) and random coil components virtually disappear. In TFE, the spectrum is dominated by the alpha-helical component (1654 cm(-1)). The band at 1662 cm(-1) shifts to 1670 cm(-1), and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm(-1), also present in TFE, has been assigned to 3(10) helical structure. The amide I' band of the complexes with the DNA retains the components that were attributed to 3(10) helix and the TPKK turn. In the complexes with the DNA, the alpha-helical component observed in TFE splits into two components at 1657 cm(-1) and 1647 cm(-1). Both components are inside the spectral region of alpha-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.     

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.     

Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.     

Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).     

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(1-40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel sheet structure was also evident. Furthermore, the peak at 1540 cm(- 1) also shifted to 1527 (1529) cm(-1) in amide II band. The former was assigned to the combination of alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C.     

Fourier transform infrared spectroscopic study on the Ca2+ -bound coordination structures of synthetic peptide analogues of the calcium-binding site III of troponin C

The coordination structures of Ca(2+) ion bound to synthetic peptide analogues of the calcium-binding site III of rabbit skeletal muscle troponin C (TnC) were investigated by Fourier transform infrared (FTIR) spectroscopy. The region of the COO(-) antisymmetric stretching vibration provides information about the coordination modes of a COO(-) group to a metal ion. The 34-residue peptide corresponding to the EF hand motif (helix-loop-helix) showed a band at 1552 cm(-1) in the Ca(2+)-loaded state, indicating that the side-chain COO(-) group of Glu at the 12th position serves as a ligand for Ca(2+) in the bidentate coordination mode. On the other hand, the 13-residue peptide (Ac-DRDADGYIDAEEL-NH(2)) containing the Ca(2+)-binding site III (DRDADGYIDAEE) did not show such spectral patterns in the Ca(2+)-loaded state, meaning that shorter synthetic peptide corresponding to the site III has less or no affinity for Ca(2+). It was found that the 17-residue peptide (Ac-DRDADGYIDAEELAEIF-NH(2)) is the minimum peptide necessary for the interaction of side-chain COO(-)of Glu at the 12th position with Ca(2+) in the bidentate coordination mode. We discuss the relationship between the amino acid length of synthetic peptide analogues and the formation of Ca(2+)-bound coordination structure.     

Pressure-induced unfolding/refolding of ribonuclease A: static and kinetic Fourier transform infrared spectroscopy study

In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.     

Abeta(1-28) fragment of the amyloid peptide predominantly adopts a polyproline II conformation in an acidic solution

To structurally characterize the nonaggregated state of the amyloid beta peptide, which assembles into the hallmark fibrils of Alzheimer disease, we investigated the conformation of the N-terminal extracellular peptide fragment Abeta(1-28) in D(2)O at acidic pD by utilizing combined FTIR and isotropic and anisotropic Raman spectra measured between 1550 and 1750 cm(-1). Peptide aggregation is avoided under the conditions chosen. The amide I' band was found to exhibit a significant noncoincidence effect in that the first moment of the anisotropic Raman and of the IR band profile appears red-shifted from that of the isotropic Raman scattering. A simulation based on a coupled oscillator model involving all 27 amide I' modes of the peptide reveals that the peptide adopts a predominantly polyproline II conformation. Our results are inconsistent with the notion that the monomeric form of Abeta(1-28) is a totally disordered, random-coil structure. Generally, they underscore the notion that polyproline II is a characteristic motif of the unfolded state of proteins and peptides.     

Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy

A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.     

Isotope-labeled vibrational circular dichroism studies of calmodulin and its interactions with ligands

In this work we have studied ligand-induced secondary structure changes in the small calcium regulatory protein calmodulin (CaM) using vibrational circular dichroism (VCD) spectroscopy. We find that, due to its chiral sensitivity, VCD spectroscopy has increased ability over IR spectroscopy to detect changes in the structure and flexibility of secondary structure elements upon ligand binding. Moreover, we demonstrate that the uniform isotope labeling of CaM with (13)C shifts its amide I' VCD band by about approximately 43 cm(-1) to lower wavenumbers, which opens up a spectral window to simultaneously visualize a bound target protein. Therefore this study also provides the first example of how isotope labeling enables protein-protein interactions to be studied by VCD with good separation of the signals for both isotope-labeled and unlabeled proteins.     

Frequency analysis of infrared absorption and vibrational circular dichroism of proteins in D2O solution.

The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add sign information to the frequency information so that features occurring in the overlapping frequency ranges that might arise from different secondary structures can be better discriminated. From this comparison, it is clear that correlation just of the frequency of a given IR transition to secondary structure can lead to a nonunique solution. Different sign patterns were identified for correlated groups of globular proteins in restricted frequency ranges that have been previously assigned to defined secondary structural elements. Hence, different secondary structural elements must contribute band components to a given frequency range.     

Changes in secondary structure and salt links of cytochrome P-450cam induced by photoreduction: a Fourier transform infrared spectroscopic study

Tris(2,2'-bipyridyl)ruthenium(II) was used as a light-induced artificial electron donor for the transfer of the first electron to cytochrome P-450(cam) bound with (1R)-camphor and camphane substrates, and in the substrate-free form. Fourier transform infrared spectroscopy was used to detect changes of the amide I' band and the CO ligand stretch vibration of heme-bound carbon monoxide associated with the heme redox transition. The reduced-minus-oxidized difference spectra show that not only the heme group but also the protein backbone and individual amino acid side chains were affected by the redox transition. Observed secondary structure changes were almost identical for (1R)-camphor-bound and camphane-bound cytochrome P-450(cam), with a remarkable negative signal at 1724.3 cm(-)(1) and a positive signal at 1716.0 cm(-)(1). These signals were not observed in substrate-free P-450(cam). On the basis of known crystallographic data, we assign these signals to a change of hydrogen bonds of a salt link between Arg112, His355, and the heme 6-propionic group.     

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.     

FT-IR Microspectroscopy of Rat Ear Cartilage

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140–820 cm^-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of –SO₃^- groups at 1064 cm^-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of –SO₃^- groups (1236–1225 cm^-1) overlapped with that of amide III bands, it is not recommended for evaluation of the –SO₃^- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027–1016 cm^-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.     

Comparative fourier transform infrared and circular dichroism spectroscopic analysis of alpha1-proteinase inhibitor and ovalbumin in aqueous solution

Alpha1-proteinase inhibitor (alpha1Pi) and ovalbumin are both members of the serpin superfamily. They share about a 30% sequence identity and exhibit great similarity in their three-dimensional structures. However, no apparent functional relationship has been found between the two proteins. Unlike alpha1Pi, ovalbumin shows no inhibitory effect to serine proteases. To see whether or not a conformational factor(s) may contribute to the functional difference, we carried out comparative analysis of the two proteins' secondary structure, thermal stability, and H-D exchange using FT-IR and CD spectroscopy. FT-IR analysis reveals significant differences in the amide I spectral patterns of the two proteins. Upon thermal denaturation, both proteins exhibit a strong low-wavenumber beta-sheet band at 1624 cm(-1) and a weak high-wavenumber beta-sheet band at 1694 cm(-1), indicative of intermolecular aggregate formation. However, the midpoint of the thermal-induced transition of alpha1Pi (approximately 55 degrees C) is 18 degrees C lower than that of ovalbumin (approximately 73 degrees C). The thermal stability analysis provides new insight into the structural changes associated with denaturation. The result of H-D exchange explains some puzzling spectral differences between the two proteins in D2O reported previously.