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.     

Structural analysis of DNA-chlorophyll complexes by Fourier transform infrared difference spectroscopy

Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios (r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration (r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 x 10(4) M-1. The pigment distributions are 60% with the backbone PO2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content (r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation.     

Photooxidation pathway of chlorophyll Z in photosystem II as Studied by Fourier transform infrared spectroscopy

The oxidation pathway of chlorophyll Z (ChlZ) in photosystem II (PSII) at cryogenic temperatures was studied by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy. To examine the involvement of redox-active beta-carotene (Car) in the pathway, two Car molecules in Mn-depleted PSII membranes of spinach were selectively bleached by illumination at 250 K in the presence of ferricyanide and silicomolybdate. Successful bleaching of Car was demonstrated by disappearance of the light-induced FTIR signals of Car+ at 1465, 1440, and 1147 cm(-1) at 80 K under an oxidative condition. Even in the Car-bleached PSII, the ChlZ+/ChlZ signal at 1713/1687 cm(-1), which is attributed to the upshift of the 9-keto C=O band of ChlZ upon its oxidation, was induced by illumination at 80 K retaining about 80% of the intensity of the control PSII sample. The concomitant appearance of shoulders at 1727/1699 cm(-1) may indicate that both of the two ChlZ molecules on the D1 and D2 sides are photooxidized. The multiphasic kinetics of formation of the ChlZ+/ChlZ signal by continuous illumination at 80 K were mostly unchanged by Car depletion, while the formation rates at 210 K were appreciably reduced in Car-bleached PSII. These results indicate that there are electron-transfer pathways from ChlZ to P680+ that do not involve Car, and they are indeed dominant at 80 K. Although the pathways via Car are mostly blocked at this temperature, the contribution of such pathways to ChlZ oxidation becomes significant at higher temperatures.     

Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy

The expression of recombinant human growth hormone (h-GH) and human interferon-alpha-2b (IFN-alpha-2b) in E. coli leads to the formation of insoluble protein aggregates or inclusion bodies (IBs). The secondary structure of these IBs, their corresponding native forms and thermal aggregates were studied by Fourier Transform Infrared (FT-IR) spectroscopy and microspectroscopy. It was demonstrated that residual native-like structures were maintained within IBs at different extents depending on the level of expression, with possible implications in biotechnology. Furthermore, comparison between infrared spectra of thermal aggregates and IBs suggests new insights on the structure of protein aggregates.     

Structural stability of the PsbQ protein of higher plant photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.     

Iron coordination in photosystem II: interaction between bicarbonate and the QB pocket studied by Fourier transform infrared spectroscopy

The non heme iron environment of photosystem II is studied by light-induced infrared spectroscopy. A conclusion of previous work [Hienerwadel, R., and Berthomieu, C. (1995) Biochemistry 34, 16288-16297] is that bicarbonate is a bidendate ligand of the reduced iron and a monodentate ligand in the Fe(3+) state. In this work, the effects of bicarbonate replacement with lactate, glycolate, and glyoxylate, and of o-phenanthroline binding are investigated to determine the specific interactions of bicarbonate with the protein. Fe(2+)/Fe(3+) FTIR spectra recorded with (12)C- and (13)C(1)-labeled lactate indicate that lactate displaces bicarbonate by direct binding to the iron through one carboxylate oxygen and the hydroxyl group in both the Fe(2+) and Fe(3+) states. This different binding mode with respect to bicarbonate could explain the lower midpoint of the iron couple observed in the presence of this anion [Deligiannakis, Y., Petrouleas, V., and Diner, B. A. (1994) Biochim. Biophys. Acta 1188, 260-270]. In agreement with the -60 mV/pH unit dependence of the iron midpoint potential in the presence of bicarbonate, the proton release upon iron oxidation by photosystem II is directly measured to 0.95 +/- 0.05 by the comparison of infrared signals of phosphate buffer and ferrocyanide modes. This accurate method may be applied to the study of other redox reactions in proteins. The pH dependence of the iron couple is proposed to reflect the deprotonation of D1His215, a putative iron ligand located at the Q(B) pocket, since the signal at 1094 cm(-1) assigned to the nu(C-N) mode of a histidinate ligand in the Fe(3+) state is not observed in the presence of o-phenanthroline. Specific regulation of the pK(a) of D1His215 by bicarbonate is inferred from the absence of the band at 1094 cm(-1) in Fe(2+)/Fe(3+) spectra recorded with glycolate, glyoxylate, or lactate. A broad positive continuum, maximum at approximately 2550 cm(-1), observed in the presence of bicarbonate, but absent with o-phenanthroline or lactate, glycolate, and glyoxylate, indicates a hydrogen bond network from the non heme iron toward the Q(B) pocket involving bicarbonate and His D1-215. Proton release of about 1, measured upon iron oxidation at pH 6 with the latter anions, points to a proton release mechanism different from that involved in the presence of bicarbonate.     

Quantitative analysis of cellulose nitrates by Fourier transform infrared spectroscopy

Quantitative express analysis of nitrogen content in cellulose nitrates by Fourier transform infrared spectroscopy has been developed. The slope of the dependence of the ratio of the band intensity (and area) to sample weight in a tablet, on the nitrogen content in a sample was used to find the reduced extinction coefficients for quantitative analysis of nitrogen content in cellulose nitrate samples by IR spectroscopy. The results were compared with the nitrogen content values in the same samples determined by the ferrosulfate method.     

Protein secondary structure of the isolated photosystem II reaction center and conformational changes studied by Fourier transform infrared spectroscopy.

The secondary structure of the photosystem II (PSII) reaction center isolated from pea chloroplasts has been characterized by Fourier transform infrared (FTIR) spectroscopy. Spectra were recorded in aqueous buffers containing H2O or D2O; the detergent present for most measurements was dodecyl maltoside. The broad amide I and amide II bands were analyzed by using second-derivative and deconvolution procedures. Absorption bands were assigned to the presence of alpha-helices, beta-sheets, turns, or random structure. Quantitative analysis revealed that this complex contained a high proportion of alpha-helices (67%) and some antiparallel beta-sheets (9%) and turns (11%). An irreversible decrease in the intensity of the band associated with the alpha-helices occurs upon exposure of the isolated PSII reaction center to bright illumination. This loss of alpha-helical content gave rise to an increase in other secondary structures, particularly beta-sheets. After similar pretreatment with light, sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals lower mobility and solubility of constituent D1 and D2 polypeptides of the PSII reaction center. Some degradation of these polypeptides also occurs. In contrast, there is no change in the mobility of the two subunits of cytochrome b559. In the absence of illumination, the PSII reaction center exchanged into dodecyl maltoside shows good thermal stability as compared with samples in Triton X-100. Only at a temperature of about 60 degrees C do spectral changes take place that are indicative of denaturation.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy

An overview of the application of Fourier transform infrared spectroscopy for the analysis of the structure of proteins and protein-ligand recognition is given. The principle of the technique and of the spectra analysis is demonstrated. Spectral signal assignments to vibrational modes of the peptide chromophore, amino acid side chains, cofactors and metal ligands are summarized. Several examples for protein-ligand recognition are discussed. A particular focus is heme proteins and, as an example, studies of cytochrome P450 are reviewed. Fourier transform infrared spectroscopy in combination with the various techniques such as time-resolved and low-temperature methods, site-directed mutagenesis and isotope labeling is a helpful approach to studying protein-ligand recognition.     

Molecular interactions of the redox-active accessory chlorophyll on the electron-donor side of photosystem II as studied by Fourier transform infrared spectroscopy

A Fourier transform infrared (FTIR) difference spectrum upon photooxidation of the accessory chlorophyll (Chl_z) of photosystem II (PS II) was obtained at 210 K with Mn-depleted PS II membranes in the presence of fericyanide and silicomolybdate. The observed Chl_z⁺/Chl_z spectrum showed two differential bands at 1747/1736 and 1714/1684 cm⁻. The former was assigned to the free carbomethoxy C = 0 and the latter to the keto C = 0 that is hydrogen-bonded or in a highly polar environment. Also, the negative 1614 cm⁻ band assignable to the macrocycle mode indicated 5-coordination of the central Mg. The negative 1660 cm⁻¹ band, possibly due to the strongly hydrogen-bonded keto C = 0, may suggest oxidation of one more Chl_z, although an alternative assignment, the amide I mode of proteins perturbed by Chl_z oxidation, is also possible.     

Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies

Two pharmacologically similar but antigenically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation. Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the -helix contents were 27 and 20%; the -sheets were 36 and 44%, the -turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively. Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of -helix and -pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm^–1 in the amide I frequency region and bands at 1314 and 1245 cm^–1 in the amide III frequency region. The peak height ratio of 1314 and 1245 cm^–1 bands, suggests that the type A neurotoxin has slightly higher -helical content than the type E neurotoxin. These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra. Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm^–1 band and a significant increase in the height of the 1314 cm^–1 band, suggesting increases in the -helical contents of the proteins. The changes were more in the type A than in the type E neurotoxin. The changes were reversible upon reexposure of the proteins to the aqueous buffer. Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent. The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.     

Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy

Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.     

S-state dependent Fourier transform infrared difference spectra for the photosystem II oxygen evolving complex

Vibrational spectroscopy provides a means to investigate molecular interactions within the active site of an enzyme. We have applied difference FTIR spectroscopy coupled with a flash turnover protocol of photosystem II (PSII) to study the oxygen evolving complex (OEC). Our data show two overlapping oscillatory patterns as the sample is flashed through the four-step S-state cycle that produces O(2) from two H(2)O molecules. The first oscillation pattern of the spectra shows a four-flash period four oscillation and reveals a number of new vibrational modes for each S-state transition, indicative of unique structural changes involved in the formation of each S-state. Importantly, the first and second flash difference spectra are reproduced in the 1800-1200 cm(-)(1) spectral region by the fifth and sixth flash difference spectra, respectively. The second oscillation pattern observed is a four-flash, period-two oscillation associated with changes primarily to the amide I and II modes and reports on changes in sign of these modes that alternate 0:0:1:1 during S-state advance. This four-flash, period-two oscillation undergoes sign inversion that alternates during the S(1)-to-S(2) and S(3)-to-S(0) transitions. Underlying this four-flash period two is a small-scale change in protein secondary structure in the PSII complex that is directly related to S-state advance. These oscillation patterns and their relationships with other PSII phenomena are discussed, and future work can initiate more detailed vibrational FTIR studies for the S-state transitions providing spectral assignments and further structural and mechanistic insight into the photosynthetic water oxidation reaction.     

Herbicide effect on the hydrogen-bonding interaction of the primary quinone electron acceptor QA in photosystem II as studied by Fourier transform infrared spectroscopy

The redox potential of Q(A) in photosystem II (PSII) is known to be lower by approximately 100 mV in the presence of phenolic herbicides compared with the presence of DCMU-type herbicides. In this study, the structural basis underlying the herbicide effects on the Q(A) redox potential was studied using Fourier transform infrared (FTIR) spectroscopy. Light-induced Q(A)(-)/Q(A) FTIR difference spectra of Mn-depleted PSII membranes in the presence of DCMU, atrazine, terbutryn, and bromacil showed a strong CO stretching peak of Q(A)(-) at 1,479 cm(-1), while binding of phenolic herbicides, bromoxynil and ioxynil, induced a small but clear downshift by approximately 1 cm(-1). The CO peak positions and the small frequency difference were reproduced in the S(2)Q(A)(-)/S(1)Q(A) spectra of oxygen-evolving PSII membranes with DCMU and bromoxynil. The relationship of the CO frequency with herbicide species correlated well with that of the peak temperatures of thermoluminescence due to S(2)Q(A)(-) recombination. Density functional theory calculations of model hydrogen-bonded complexes of plastoquinone radical anion showed that the small shift of the CO frequency is consistent with a change in the hydrogen-bond structure most likely as a change in its strength. The Q(A)(-)/Q(A) spectra in the presence of bromoxynil, and ioxynil, which bear a nitrile group in the phenolic ring, also showed CN stretching bands around 2,210 cm(-1). Comparison with the CN frequencies of bromoxynil in solutions suggested that the phenolic herbicides take a phenotate anion form in the Q(B) pocket. It was proposed that interaction of the phenolic C-O(-) with D1-His215 changes the strength of the hydrogen bond between the CO of Q(A) with D2-His214 via the iron-histidine bridge, causing the decrease in the Q(A) redox potential.     

Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of primary reactions in photosystem I and photosystem II

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been applied for the first time to primary reactions in green plant photosynthesis. Photooxidation of the primary electron donor (P700) in photosystem I-enriched particles as well as in thylakoids, and photoreduction of the pheophytin (Pheo) intermediary electron acceptor in photosystem II-enriched particles, have led to reproducible difference spectra. In the spectral range investigated (between 1800 and 1000 cm⁻¹) several bands are tentatively attributed to changes in intensity and position of the keto and ester carbonyl vibrations of the chlorophyll or Pheo molecule(s) involved. For some of these groups, possible interpretations in terms of changes of their environment or type of bonding to the protein are given. The intensity of the differential features in the amide I and amide II spectral region allows the exclusion of the eventuality of large protein conformational changes occurring upon primary charge separation.     

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR₁C (1–77) and TR₂C (78–148). Experiments were done in H₂O, D₂O and D₂O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca²⁺-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR₁C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D20 was markedly different (1642 cm^–1 for TR₁C versus 1646/1648 cm^–1 for Ca ²⁺ and apo-TR₂C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of -helices with slightly different hydrogen bonding patterns.