Inelastic neutron-scattering study of the proton transfer dynamics in polyglycine I at 20 K

Inelastic neutron-scattering (INS) spectra of three isotopic derivatives of polyglycine I (-COCH2NH-)n, (-COCD2NH-)n, and (-COCH2ND-)n at 20 K are presented from 30 to 4000 cm(-1). The band frequencies are compared to those observed in the infrared and Raman. Assignments in terms of group vibrations are proposed. These mostly resemble previous assignment schemes, except for the amide bands. The INS intensities reveal that the proton dynamics for the (N)H proton are totally different from those proposed previously. They are independent of the molecular frame and the valence bond approach is not consistent with observation. A phenomenological approach is proposed in terms of localized modes. The calculated intensities reveal that the (N)H stretching mode has two components at approximately 1377 and 1553 cm(-1). This is a dramatic change compared to all former assignments at approximately 3280 cm(-1) based on infrared and Raman data. These proton-dynamics are associated with a weakening of the NH bond due to the ionic character of the hydrogen bond (N(delta-)...H+...O(delta'-)) and proton transfer. The infrared and Raman spectra are re-examined and a new assignment scheme is proposed for the amide bands; the amide A and B bands are re-assigned to the overtones of the stretching modes. A symmetric double-minimum potential for the proton is consistent with all the observations.     

Origin of enhanced VCD in amyloid fibril spectra: Effect of deuteriation and pH

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D₂O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.     

Structure and dynamics of the DNA-binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy

The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an alpha-helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain beta-stranded structure in excess (eight +/- two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457 cm(-1)). The Ala marker undergoes a substantial shift (908 --> 893 cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the alpha-helical core exhibits robust resistance to exchange. A quantitative study of NH --> ND exchange exploiting newly identified amide II' markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1)) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins.     

Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure

As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.     

Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.     

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and alpha1-acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679-688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289-300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils.     

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

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

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

Investigations of the supramolecular structure of individual diphenylalanine nano- and microtubes by polarized Raman microspectroscopy

Polarized Raman microspectroscopy and atomic force microscopy were used to obtain quantitative information regarding the molecular structure of individual diphenylalanine (FF) nano- and microtubes. The frequencies of the Raman spectral bands corresponding to the amide I (1690 cm(-1)) and amide III (1249 cm(-1)) indicated that the FF-molecules interact by hydrogen bonding at the N-H and not at the C═O sites. The calculated mean orientation angles of the principal axes of the Raman tensors (PARTs) obtained from the polarized Raman spectral measurements were 41 ± 4° for the amide I and 59 ± 5° for amide III. On the basis of the orientation of the PART for the amide I mode, it was found that the C═O bond is oriented at an angle of 8 ± 4° to the tube axis. These values did not vary significantly with the diameter of the tubes (range 400-1700 nm) and were in agreement with the molecular structure proposed previously for larger crystalline specimens.     

Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.     

FTIRS in H2O demonstrates that collagen monomers undergo a conformational transition prior to thermal self-assembly in vitro

The assembly of type I collagen molecules into native fibrils can be accomplished in vitro in solutions at physiological ionic strength and pH by raising the temperature above 30 degrees C. The thermal self-assembly reaction exhibits a distinct lag phase. This lag phase has been proposed to be evidence for a conformational transition in the monomer. Fourier transform infrared spectroscopy (FTIRS) is a very sensitive probe of the H-bonded states within the triple helix. The carbonyl group spectrum (amide I, 1700-1600 cm-1) has been investigated in collagen/H2O solutions at 1 mg/mL under self-assembly conditions from 4 to 34 degrees C and, in the same range, at a higher ionic strength where self-assembly does not occur. The deconvoluted spectra show three very clear bands at approximately 1660, 1644, and 1630 cm-1. These bands vary in both frequency maxima and relative intensity over the temperature range examined. Spectra were also obtained in the amide II and III regions. Spectral changes were evident in the 22-26 degrees C range, under fibril-forming conditions, which lead to the hypothesis that the triple helix of the semiflexible collagen molecule is actually perfected during the lag phase, facilitating nucleation and intermolecular interaction. Further spectral changes after fibrils do form show that the molecules are once again distorted as they are bent to fit within the fibrils.     

Secondary structure determination in proteins from deep (192-223-nm) ultraviolet Raman spectroscopy

Raman intensities obtained with UV laser excitation at 223, 218, 204, 200, and 192 nm are reported for the amide I, II, III, and II' bands of random-coil polylysine. The excitation profiles show enhancement via the pi-pi electronic transition, at approximately 190 nm. Enhancement for amide I is weak, however, and most of the intensity can be accounted for by preresonance with a deeper UV transition at approximately 165 nm. The amide II' band dominates the spectrum in D2O, consistent with the suggestion that the main distortion coordinate in the pi-pi excited state is the stretching of the C-N peptide bond. Amide II intensities with 200- and 192-nm excitation are reported for several proteins. The previously reported negative linear correlation with alpha-helix content (due to Raman hypochromism in the alpha-helices) is found not to apply to proteins with high beta-sheet content when the excitation wavelength is 200 nm. Much higher intensities are seen for these proteins and are attributed to a red shift of the pi-pi absorption for the beta-structure. A linear correlation with alpha-helix content is found for excitation of 192 nm, which corresponds to an isosbestic point of the beta-sheet and random-coil absorption bands. Characteristic amide II Raman cross sections are derived for alpha-helical, beta-sheet, and random-coil elements and are used to determine secondary structure for alpha 1- and beta-purothionin, by use of amide II intensities with 200- and 192-nm excitation. The results are in good agreement with a previous determination based on amide I band deconvolution in off-resonance Raman spectra.     

Effect of different anti-Abeta antibodies on Abeta fibrillogenesis as assessed by atomic force microscopy

Extensive data suggest that the conversion of the amyloid-beta (Abeta) peptide from soluble to insoluble forms is a key factor in the pathogenesis of Alzheimer's disease (AD). In recent years, atomic force microscopy (AFM) has provided useful insights into the physicochemical processes involving Abeta morphology, and it can now be used to explore factors that either inhibit or promote fibrillogenesis. We used ex situ AFM to explore the impact of anti-Abeta antibodies directed against different domains of Abeta on fibril formation. For the AFM studies, two monoclonal antibodies (m3D6 and m266.2) were incubated in solution with Abeta(1-42) with a molar ratio of 1:10 (antibody to Abeta) over several days. Fibril formation was analyzed quantitatively by determining the number of fibrils per microm(2) and by aggregate size analysis. m3D6, which is directed against an N-terminal domain of Abeta (amino acid residues 1-5) slowed down fibril formation. However, m266.2, which is directed against the central domain of Abeta (amino acid residues 13-28) appeared to completely prevent the formation of fibrils over the course of the experiment. Inhibition of fibril formation by both antibodies was also confirmed by thioflavin-T (ThT) fluorescence experiments carried out with Abeta(1-40) incubated for five days. However, unlike AFM results, ThT did not differentiate between the samples incubated with m3D6 versus m266.2. These results indicate that AFM can be not only reliably used to study the effect of different molecules on Abeta aggregation, but that it can provide additional information such as the role of epitope specificity of antibodies as potential inhibitors of fibril formation.     

Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide

We report investigations of the morphology and molecular structure of amyloid fibrils comprised of residues 10-40 of the Alzheimer's beta-amyloid peptide (Abeta(10-40)), prepared under various solution conditions and degrees of agitation. Omission of residues 1-9 from the full-length Alzheimer's beta-amyloid peptide (Abeta(1-40)) did not prevent the peptide from forming amyloid fibrils or eliminate fibril polymorphism. These results are consistent with residues 1-9 being disordered in Abeta(1-40) fibrils, and show that fibril polymorphism is not a consequence of disorder in residues 1-9. Fibril morphology was analyzed by atomic force and electron microscopy, and secondary structure and inter-side-chain proximity were probed using solid-state NMR. Abeta(1-40) fibrils were found to be structurally compatible with Abeta(10-40): Abeta(1-40) fibril fragments were used to seed the growth of Abeta(10-40) fibrils, with propagation of fibril morphology and molecular structure. In addition, comparison of lyophilized and hydrated fibril samples revealed no effect of hydration on molecular structure, indicating that Abeta(10-40) fibrils are unlikely to contain bulk water.     

Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.