FTIR spectra of solid poly-l-lysine in the stretching NH mode range

Three bands at 3270 cm(-1), 3200 cm(-1) and 3030 cm(-1) are found in the IR stretching proton (nu(1)) mode spectral range in spectra of solid poly-l-lysine (PLL). Strong quantitative changes of these bands are observed in samples dried from water solutions with different pH. The narrow band at 3270 cm(-1), which is strong in the spectrum of PLL precipitated from pH=12 alkaline medium, is assigned to the nu(1) peptide proton mode of NH-CO (amide A) of the beta-sheet structure type. The band at 3200 cm(-1), which is intensified in PLL precipitated from pH=1 acidic medium, relates to the nu(1) peptide mode in the random coil structure. The band at 3030 cm(-1), whose peak intensity increases two-fold in going from alkaline to acidic medium, is assigned to the nu(1) modes of protonated NH(3)(+) side chain groups. The frequencies of all bands were used for estimating H-bond energy relying on an empirical correlation between this property and the red shift of the nu(1) band. The enthalpy of the secondary structure transition from beta-sheet to the random coil, which is observed in PLL at the change of pH from 11 to 1 amounts to 4.7 kJ mol(-1).     

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.     

Structure and stability of the CuII complexes with tandem repeats of the chicken prion

Prion protein (PrP) misfolding is one of the pivotal issues in understanding the rudiments of neurodegenerative disorders. The conformational change of mammalian cellular PrP to scrapie PrP is caused by an unknown agent, but there is reasonable evidence supporting the key role of copper ions in this process. The structure of the avian PrP was found to be very similar to the mammalian protein, although there is only 30% homology in the secondary structure. This work shows that copper ions are very effectively bound by hexarepeat fragments of chicken prion protein, although not as effectively as it was found in the case of mammalian protein. By means of potentiometric and spectroscopic techniques (nuclear magnetic resonance, circular dichroism, UV-vis, and electronic paramagnetic resonance), it was shown that Cu(II) ions coordinate to the chicken PrP hexapeptide domain in physiological pH via imidazole nitrogen donors of His residue(s). The binding pattern changes the structure of peptide involved, indicating a possible impact of Cu(II) ions in the biology and pathology of nonmammalian PrP, which could be similar to that found for mammalian PrP. The present study shows that, similar to the human prion octapeptide repeats, chicken prion hexapeptide repeats might bind copper ions in two different ways, depending on the number of repeats and metal/ligand molar ratio: (i) an intra-repeat coordination mode in which copper ion is chelated by His imidazole and deprotonated amide nitrogen in monomeric peptide and (ii) an inter-repeat coordination mode in which a polymeric peptide ligand (dimer and trimer) forms polyimidazole complexes that are very stable at physiological pH. Two proline residues inserted into the hexapeptide unit have a critical impact on the metal-binding ability.     

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.     

Water structural changes involved in the activation process of photoactive yellow protein

Fourier transform infrared (FTIR) spectroscopy was applied to the blue-light photoreceptor photoactive yellow protein (PYP) to investigate water structural changes possibly involved in the photocycle of PYP. Photointermediates were stabilized at low temperature, and difference IR spectra were obtained between intermediate states and the original state of PYP (pG). Water structural changes were never observed in the >3570 cm(-)(1) region for the intermediates stabilized at 77-250 K, such as the red-shifted pR and blue-shifted pB intermediates. In contrast, a negative band was observed at 3658 cm(-)(1) in the pB minus pG spectrum at 295 K, which shifts to 3648 cm(-)(1) upon hydration with H(2)(18)O. The high frequency of the O-H stretch of water indicates that the water O-H group does not form hydrogen bonds in pG, and newly forms these upon pB formation at 295 K, but not at 250 K. Among 92 water molecules in the crystal structure of PYP, only 1 water molecule, water-200, is present in a hydrophobic core inside the protein. The amide N-H of Gly-7 and the imidazole nitrogen atom of His-108 are its possible hydrogen-bonding partners, indicating that one O-H group of water-200 is free to form an additional hydrogen bond. The water band at 3658 cm(-)(1) was indeed diminished in the H108F protein, which strongly suggests that the water band originates from water-200. Structural changes of amide bands in pB were much greater in the wild-type protein at 295 K than at 250 K or in the H108F protein at 295 K. The position of water-200 is >15 A remote from the chromophore. Virtually no structural changes were reported for regions larger than a few angstroms away from the chromophore, in the time-resolved X-ray crystallography experiments on pB. On the basis of the present results, as well as other spectroscopic observations, we conclude that water-200 (buried in a hydrophobic core in pG) is exposed to the aqueous phase upon formation of pB in solution. In neither crystalline PYP nor at low temperature is this structural transition observed, presumably because of the restrictions on global structural changes in the protein under these conditions.     

Two-dimensional near-IR correlation spectroscopy study of molten globule-like state of ovalbumin in acidic pH region: simultaneous changes in hydration and secondary structure

The molten globule-like states of ovalbumin (OVA) in acid aqueous solutions are investigated by generalized two-dimensional (2D) Fourier transform near-IR (FT-NIR) correlation spectroscopy. This new method allows us to explore the changes in hydration and the secondary structure simultaneously. FT-NIR spectra are measured for OVA aqueous solutions with concentrations of 1, 2, 3, 4, and 5 wt % over a pH range of 2.4-5.4. Concentration-perturbed 2D correlation spectra are calculated for the spectra in the 4850-4200 and 7500-5350 cm(-1) regions at different pH values. The 2D NIR synchronous spectrum in the 4850-4200 cm(-1) region shows a significant change upon going from pH 5.4 to 3.6. An autopeak at 4265 cm(-1) that is due to a combination of a symmetric CH(2) stretching mode and a CH(2) bending mode of side chains seen at pH 5.0 disappears completely in the synchronous spectrum at pH 3.6. This suggests that some amino acid residues of OVA are subjected to microenvironmental changes with decreasing pH. More remarkable changes are observed in the synchronous spectra at pHs below 2.8. A band near 4600 cm(-1) arising from a combination of amide B and amide II modes (amide B/II) shifts downward with considerable broadening between pH 3.0 and 2.4, suggesting that the strength of the hydrogen bonds of amide groups of OVA changes significantly. The synchronous and asynchronous spectra in the 4850-4200 cm(-1) region show that the intensities of the bands attributable to amide groups and side chains of OVA and that of the band near 4800 cm(-1) arising from water change in phase with the increase in the concentration above pH 2.8, but they vary out of phase below pH 2.8. The 2D synchronous map in the 7500-5350 cm(-1) region also shows marked changes upon going from pH 2.8 to 2.6. A broad autopeak at around 6950 cm(-1) assigned to free water and bound water with weak hydrogen bonds becomes very weak in the synchronous spectrum at pH 2.6, while broad autopeaks around 6450 cm(-1) suddenly appear that are due to bound water with several hydrogen bonds and the first overtone of an NH stretching mode of the amide groups of OVA. Therefore, it is very likely that protein hydration and the hydrogen bonds of amide groups change simultaneously in a narrow pH region of 2.8-2.6. It is probably that below pH 2.6 the protein assumes a molten globule-like state in which the whole molecule is very flexible, and side chains (but not the backbone chain) fluctuate significantly.     

De novo design of a copper(II)-binding helix-turn-helix chimera: the prion octarepeat motif in a new context

A chimeric Cu-binding peptide has been designed on the basis of a turn substitution of the prion (PrP) octarepeat Cu-binding site into the engrailed homeodomain helix-turn-helix motif (HTH). This system is a model for the investigation of a single PrP Cu-binding site in a defined protein context. The 28-mer Cu-HTH peptide P7 spectroscopically mimics the PrP octarepeat (P7 = TERRRQQLSHGGGWGEAQIKIWFQNKRA). The Cu(II)-binding affinity of P7 was determined by ESI-MS and tryptophan fluorescence titrations to be K(d) = 2.5 +/- 0.7 microM at pH = 7.0. The quenching of fluorescence of the Trp within the binding loop (underlined above) is pH dependent and highly specific for Cu(II). No Trp quenching was observed in the presence of divalent Zn, Mn, Co, Ni, or Ca ions, and ESI-MS titrations confirmed that these divalent ions do not appreciably bind to P7. The EPR spectrum of Cu(II)-P7 shows that the Cu environment is axial and consistent with 6-coordinate N(3)O(H(2)O)(2) or N(4)(H(2)O)(2) coordination (A( parallel) = 172 x10(-)(4) cm(-)(1); g( parallel) = 2.27), very similar to that of the PrP octarepeat itself. Also like PrP, circular dichroism studies show that apo P7 is predominantly disordered in solution, and the structure is slightly enhanced by Cu binding. These data show the Cu-PrP HTH peptide reproduces the Cu-binding behavior of a single PrP octarepeat in a new context.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.     

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.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

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.     

Phosphoenzyme conversion of the sarcoplasmic reticulum Ca(2+)-ATPase. Molecular interpretation of infrared difference spectra.

Time-resolved Fourier transform infrared difference spectra of the phosphoenzyme conversion and Ca(2+) release reaction (Ca(2)E(1)-P --> E(2)-P) of the sarcoplasmic reticulum Ca(2+)-ATPase were recorded at pH 7 and 1 degrees C in H(2)O and (2)H(2)O. In the amide I spectral region, the spectra indicate backbone conformational changes preserving conformational changes of the preceding phosphorylation step. beta-sheet or turn structures (band at 1685 cm(-1)) and alpha-helical structures (band at 1653 cm(-1)) seem to be involved. Spectra of the model compound EDTA for Ca(2+) chelation indicate the assignment of bands at 1570, 1554, 1411 and 1399 cm(-1) to Ca(2+) chelating Asp and Glu carboxylate groups partially shielded from the aqueous environment. In addition, an E(2)-P band at 1638 cm(-1) has been tentatively assigned to a carboxylate group in a special environment. A Tyr residue seems to be involved in the reaction (band at 1517 cm(-1) in H(2)O and 1515 cm(-1) in (2)H(2)O). A band at 1192 cm(-1) was shown by isotopic replacement in the gamma-phosphate of ATP to originate from the E(2)-P phosphate group. This is a clear indication that the immediate environment of the phosphoenzyme phosphate group changes in the conversion reaction, altering phosphate geometry and/or electron distribution.     

Assembly of the full-length recombinant mouse prion protein I. Formation of soluble oligomers

The conversion of a monomeric alpha-helix-rich isoform to multimeric beta-sheet-rich isoforms is a prominent feature of the conversion between PrP(C) and PrP(SC). We mimicked this process in vitro by exposing an unglycosylated recombinant form of the full-length mouse prion protein ((Mo)PrP(23-231)) to an acidic pH, at 37 degrees C, and we monitored the kinetics of conformational change and assembly. In these conditions, monomeric (Mo)PrP(23-231) converts slowly to two ensembles of soluble oligomers that are separated by size exclusion chromatography. The larger oligomers (I) are unstable, and their formation involves almost no change in secondary structure content. The smaller oligomers (II) form stable spherical or annular particles containing between 8 and 15 monomers as determined by multi-angle laser light scattering (MALLS). Their formation is concomitant with the main, thought limited, change in the secondary structure content (10%) seen by Fourier Transform Infrared (FTIR) spectroscopy. Even if these oligomers conserve a large part of the secondary structure of monomeric PrP, they exhibit amyloid features with the appearance of intermolecular beta-structure as revealed by the appearance of an IR band below 1620 cm(-1).     

NMR studies of amyloid beta-peptides: proton assignments, secondary structure, and mechanism of an alpha-helix----beta-sheet conversion for a homologous, 28-residue, N-terminal fragment.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.     

Structural differences between TSEs strains investigated by FT-IR spectroscopy

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.     

Fourier transform infrared evidence of proton uptake by glutamate L212 upon reduction of the secondary quinone QB in the photosynthetic reaction center from Rhodobacter capsulatus

The photoreduction of the secondary quinone Q(B) in native reaction centers (RCs) of Rhodobacter capsulatus and in RCs from the GluL212 --> Gln and GluL212 --> Ala mutants has been investigated at pH 7 in (1)H(2)O and (2)H(2)O by light-induced Fourier transform infrared (FTIR) difference spectroscopy. The Q(B)(-)/Q(B) FTIR difference spectra reflect changes of quinone-protein interactions and of protonation state of carboxylic acid groups as well as reorganization of the protein upon electron transfer. Comparison of Q(B)(-)/Q(B) spectra of native and mutant RCs indicates that the interactions between Q(B) or Q(B)(-) and the protein are similar in all RCs. A differential signal at approximately 1650/1640 cm(-1), which is common to all the spectra, is associated with a movement of a peptide carbonyl or a side chain following Q(B) reduction. On the other hand, Q(B)(-)/Q(B) spectra of native and mutant RCs display several differences, notably between 1700 and 1650 cm(-1) (amide I and side chains), between 1570 and 1530 cm(-1) (amide II), and at 1728-1730 cm(-1) (protonated carboxylic acid groups). In particular, the latter region in native RCs is characterized by a main positive band at 1728 cm(-1) and a negative signal at 1739 cm(-1). In the L212 mutants, the amplitude of the positive band is strongly decreased leading to a differential signal at 1739/1730 cm(-1) that is insensitive to (1)H/(2)H isotopic exchange. In native RCs, only the 1728 cm(-1) band is affected in (2)H(2)O while the 1739 cm(-1) signal is not. The effects of the mutations and of (1)H/(2)H exchange on the Q(B)(-)/Q(B) spectra concur in the attribution of the 1728 cm(-1) band in native RCs to (partial) proton uptake by GluL212 upon the first electron transfer to Q(B), as previously observed in Rhodobacter sphaeroides RCs [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732]. More generally, strong homologies of the Q(B) to Q(B)(-) transition in the RCs from Rb. sphaeroides and Rb. capsulatus are detected by differential FTIR spectroscopy. The FTIR data are discussed in relation with the results from global proton uptake measurements and electrogenic events concomitant with the reduction of Q(B) and with a model of the Q(B) turnover in Rb. sphaeroides RCs [Mulkidjanian, A. Y. (1999) FEBS Lett. 463, 199-204].     

A role for His155 in binding of human prion peptide144-167 to immobilised prion protein

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167).     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.     

Thermal-induced changes in the secondary conformation of superoxide dismutase containing different metal ions

The thermal stability of three superoxide dismutases (SODs) with different metal ions (Mn, Cu/Zn, Fe) in the solid state was studied by a Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analyzer. The IR spectra showed a maximum peak at 1652 cm(-1) for all the native SODs in the amide I band, suggesting a predominant random coil with less alpha-helix structures. By heating each sample, a shoulder at 1631 cm(-1) in the amide I band gradually appeared from 45 degrees C for Fe SOD and from 50 degrees C for Mn SOD but another shoulder at 1639 cm(-1) appeared from 50 degrees C for Cu/Zn SOD. The peak at 1631 cm(-1) is due to the intermolecular beta-sheet structure, but the peak at 1639 cm(-1) corresponds to the major intramolecular beta-sheet with less random coil structure. This reveals that in the first heating process the transformation from random coil/alpha-helix structure to beta-sheet structure initiated from around 45-50 degrees C. There was about 16-22% compositional change resulting from that transformation. However, both additional shoulders stood there and did not restore to their original spectra even with cooling to room temperature, suggesting the denaturation and irreversible properties of the solid SODs after heating. The thermal-dependent denaturation and irreversibility of Mn SOD, Cu/Zn SOD and Fe SOD were clearly evidenced by the increase in intramolecular and intermolecular beta-sheet structure.     

Cell cycle and activation of CK2

Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1–91) and tandem repeats region (amino acids 186–283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.