Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy

The secondary structure of adenylate kinase (EC 2.7.4.3) from E. coli was investigated under various conditions using Fourier transform infrared spectroscopy. The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is alpha-helical. A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% alpha-helix, 26% beta-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures. Binding of nucleotide (e.g., ATP) to the donor site decreases the beta-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein. These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin. The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed.     

Fourier transform infrared investigation of the Escherichia coli methionine aporepressor

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.     

Study of the structure of arrestin (S-antigen) from bovine photoreceptors by FTIR spectroscopy.

Fourier transform-infrared spectroscopy has been used for the study of the secondary structure of arrestin from bovine retina rod cells. Spectra have been obtained in H2O and in D2O media. Resolution enhancement of the amide I secondary structure-sensitive overlapped component bands has been achieved by means of Fourier self-deconvolution and Fourier derivation. In order to obtain a quantitative estimation of the proportion of amino acid residues involved in each type of secondary structure, bands at the resolved frequencies have been curve-fitted to the deconvolved amide I contour by means of a least-squares best-fitting iterative program. The analysis of the results suggests that the secondary structure of arrestin comprises 56-63% of extended strands, 12-19% of turns and bends, 15% of alpha-helices and 10% of undefined and irregular segments.     

Botulinum neurotoxin type A: Structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm^–1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm^–1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% -helix, 45–49% -sheets and 22–26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.     

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.     

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.     

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 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.     

Valinomycin and its interaction with ions in organic solvents, detergents, and lipids studied by Fourier transform IR spectroscopy.

The structure of valinomycin in a range of organic solvents of varying polarity and in detergent and lipid dispersions has been studied by Fourier transform ir spectroscopy. In solvents of low polarity such as chloroform, ir spectra of valinomycin are fully consistent with the bracelet structure proposed on the basis of nmr spectroscopy, showing a single narrow amide I component attributable to the presence of beta-turns and a single band arising from nonhydrogen-bonded ester C = O groups. K+ complexation results in a downward shift in the amide I band frequency, indicating an increase in the strength of the amide hydrogen bonds, along with a shift to lower frequencies of the ester C = O absorption due to a reduction in electron density in these bonds upon complexation. Identical results were obtained with NH4+, a finding not previously reported. In solvents of both medium (CHCl3/DMSO 3:1) and high (pure DMSO) polarity, we find evidence of significant disruption of the internal hydrogen-bonding network of the peptide and the appearance of a band suggesting the presence of free amide C = O groups. In such solvents, complexation with K+ and NH4+ was not observed. The structure of valinomycin in detergent micelles resembles that in nonpolar organic solvents. However, changes were found in the amide I and ester carbonyl maxima as 2H2O penetrated the micelle which suggest significant interaction between the solvent and peptide. Complexation with K+ was reduced in cationic detergent micelles as a result of a decrease in the effective K+ concentration due to charge repulsion at the micelle surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.     

Centrin: its secondary structure in the presence and absence of cations

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.     

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.     

Interaction of alcohols with serum LDL An infrared study

The interaction of low molecular weight alcohols with low density lipoprotein (LDL) has been studied using amide I band-fitting, thermal profiling and two-dimensional infrared correlation spectroscopy (2D-IR). At 0.3 M alcohol, no changes in secondary structure are observed. In the presence of 1 M alcohol, ethanol and propanol decreases protein denaturation temperature and produces changes in the amide I thermal profiles of protein components and in the lipid bands. The 2D-IR synchronous map corresponding to protein or lipid component at 20-37 degrees C suggests differences in the presence of propanol. The asynchronous map corresponding to the lipid component indicates changes in bandwidth, compatible with a more fluid environment. In the 37-80 degrees C temperature range the thermal profile is different in the presence of propanol, both for the lipid and protein components. The results presented show that when alcohols affect the protein component, the lipid spectrum also varies pointing to an effect on the lipid-protein interaction.     

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.     

A1 reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy, with 5 mus time resolution, has been used to produce P700(+)A(1)(-)/P700A(1) FTIR difference spectra in intact photosystem I particles from Synechococcus sp. 7002 and Synechocystis sp. 6803 at 77 K. Corresponding spectra were also obtained for fully deuterated photosystem I particles from Synechococcus sp. 7002 as well as fully (15)N- and (13)C-labeled photosystem I particles from Synechocystis sp. 6803. Static P700(+)/P700 FTIR difference spectra at 77 K were also obtained for all of the unlabeled and labeled photosystem I particles. From the time-resolved and static FTIR difference spectra, A(1)(-)/A(1) FTIR difference spectra were constructed. The A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled trimeric photosystem I particles from both cyanobacterial strains are very similar. There are some mode frequency differences in spectra obtained for monomeric and trimeric PS I particles. However, the spectra can be interpreted in an identical manner, with the proposed band assignments being compatible with all of the data obtained for labeled and unlabeled photosystem I particles. In A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled photosystem I particles, negative bands are observed at 1559 and 1549-1546 cm(-)(1). These bands are assigned to amide II protein vibrations, as they downshift approximately 86 cm(-)(1) upon deuteration and approximately 13 cm(-)(1) upon (15)N labeling. Difference band features at 1674-1677(+) and 1666(-) cm(-)(1) display isotope-induced shifts that are consistent with these bands being due to amide I protein vibrations. The observed amide modes suggest alteration of the protein backbone (possibly in the vicinity of A(1)) upon A(1) reduction. A difference band at 1754(+)/1748(-) cm(-)(1) is observed in unlabeled spectra from both strains. The frequency of this difference band, as well as the observed isotope-induced shifts, indicate that this difference band is due to a 13(3) ester carbonyl group of chlorophyll a species, most likely the A(0) chlorophyll a molecule that is in close proximity to A(1). Thus A(1) reduction perturbs A(0), probably via a long-range electrostatic interaction. A negative band is observed at 1693 cm(-)(1). The isotope shifts associated with this band are consistent with this band being due to the 13(1) keto carbonyl group of chlorophyll a, again, most likely the 13(1) keto carbonyl group of the A(0) chlorophyll a that is close to A(1). Semiquinone anion bands are resolved at approximately 1495(+) and approximately 1414(+) cm(-)(1) in the A(1)(-)/A(1) FTIR difference spectra for photosystem I particles from both cyanobacterial strains. The isotope-induced shifts of these bands could suggest that the 1495(+) and 1414(+) cm(-)(1) bands are due to C-O and C-C modes of A(1)(-), respectively.     

Studies of peptides forming 3(10)- and alpha-helices and beta-bend ribbon structures in organic solution and in model biomembranes by Fourier transform infrared spectroscopy

In order to examine the potential correlation between infrared absorption spectra and 3(10)- and alpha-helices and beta-bend ribbon structures, the secondary structures of synthetic peptides known to contain pure 3(10)-helices, mixed 3(10)/alpha-helices, and pure beta-bend ribbon structures, based upon X-ray diffraction and NMR studies, have been investigated by using FTIR spectroscopy incorporating resolution-enhancement techniques. Studies of the peptides known to contain a stable 3(10)-helix in CDCl3 show the main amide I band of fully stable 3(10)-helices occurs at 1666-1662 cm-1. Resolution-enhancement methods revealed small contributions at 1681-1678 and 1646-1644 cm-1, while the amide II band occurs at 1533-1531 cm-1. Peptides known to contain both alpha- and 3(10)-helices in their structure exhibit bands characteristic of both types of conformation. Peptides known to fold into the beta-bend ribbon structure show an amide I band maximum at 1648-1645 cm-1 with the amide II band at 1538-1536 cm-1. Incorporation of these peptides into model membrane structures, e.g., DMPC vesicles, in aqueous buffer sometimes produces changes in the peptide secondary structure. Those peptides which possess a 3(10)-helical structure in CDCl3 solution change the secondary structure in DMPC vesicles to predominantly alpha-helical, plus a contribution from short, unstable 3(10)-helix and/or beta-turns. Those peptides which contain a combination of alpha- and 3(10)-helical structures in CDCl3 solution tend to retain some 3(10)-helical structure within the lipid environment, although the overall H-bonding pattern is altered. Those peptides which form a beta-bend ribbon structure appear to be largely unaffected in the membrane environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

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)     

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.     

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.