Amyloid fiber formation in human γD-Crystallin induced by UV-B photodamage.

γD-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-Crystallin. The native ß-sandwich domains are not retained upon precipitation by either mechanism. The similarities between the amyloid forming pathways when induced by either UV-B radiation or low pH suggest that the propensity for the C-terminal ß-sandwich domain to form amyloid ß-sheets determines the misfolding pathway independent of the mechanism of denaturation.

Structural and sequence analysis of the human γD-crystallin amyloid fibril core using 2D IR spectroscopy, segmental 13C labeling, and mass spectrometry.

Identifying the sequence and structural content of residues that compose the core of amyloid fibrils is important because core regions likely control the process of fibril extension and provide potential drug targets. Human γD-crystallin is an eye lens protein that aggregates into amyloid fibrils under acidic conditions. In this manuscript, we use a pepsin enzymatic digest to isolate the core of the amyloid fibrils. The sequence of the core is identified with MALDI MS/MS and its structure is probed with 2D IR spectroscopy and (13)C isotope labeling. Mass spectrometry of the digest identifies residues 80-163 as the amyloid core, which spans most of the C-terminal domain, the linker, and a small portion of the N-terminal domain. From 2D IR spectroscopy of the digested fibrils, we learn that only the C-terminal domain contributes to the amyloid ß-sheets while the N-terminal and linker residues are disordered. A comparison to the native crystal structure reveals that loops and α-helices in the native state must undergo conformational transitions to ß-strands upon aggregation. These locations may be good drug binding targets. Besides providing new information about γD-crystallin, this study demonstrates the complementarity of mass spectrometry and 2D IR spectroscopy to obtain both sequence and structure information that neither technique provides individually, which will be especially useful for samples only available in microgram quantities.

Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils.

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid ß-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the ß-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent ß-strands to each ß-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 µg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation.

Self-aggregation of a polyalanine octamer promoted by its C-terminal tyrosine and probed by a strongly enhanced vibrational circular dichroism signal.

The eight-residue alanine oligopeptide Ac-A(4)KA(2)Y-NH(2) (AKY8) was found to form amyloid-like fibrils upon incubation at room temperature in acidified aqueous solution at peptide concentrations >10 mM. The fibril solution exhibits an enhanced vibrational circular dichroism (VCD) couplet in the amide I' band region that is nearly 2 orders of magnitude larger than typical polypeptide/protein signals in this region. The UV-CD spectrum of the fibril solution shows CD in the region associated with the tyrosine side chain absorption. A similar peptide, Ac-A(4)KA(2)-NH(2) (AK7), which lacks a terminal tyrosine residue, does not aggregate. These results suggest a pivotal role for the C-terminal tyrosine residue in stabilizing the aggregation state of this peptide. It is speculated that interactions between the lysine and tyrosine side chains of consecutive strands in an antiparallel arrangement (e.g., cation-pi interactions) are responsible for the stabilization of the resulting fibrils. These results offer considerations and insight regarding the de novo design of self-assembling oligopeptides for biomedical and biotechnological applications and highlight the usefulness of VCD as a tool for probing amyloid fibril formation.

Different modes of binding of mono-, di-, and trihalogenated phenols to the hemoglobin dehaloperoxidase from Amphitrite ornata.

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.

Fiber diffraction as a screen for amyloid inhibitors.

Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimer's, Parkinson's, Huntington's, etc., as the early-stage, oligomers that form before the development of beta-conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.7 A corresponding to the hydrogen-bonding spacing between the beta-chains, and the other at approximately 10 A corresponding to the spacing between beta-pleated sheets. We report here the application of fiber x-ray diffraction to monitor these structural indicators of amyloid fiber assembly in the presence of small, aromatic molecules, some of which have been assessed by other techniques as being inhibitory. The compounds included butylated hydroxytoluene, chloramphenicol, cotinine, curcumin, diphenylalanine (FF), ethyl 3-aminobenzoate methane sulfonate, hexachlorophene, melatonin, methylpyrrolidine, morin, nicotine, phenolphthalaine, PTI-00703 (Cat's claw), pyridine, quinine, sulfadiazine, tannic acid, tetracaine, tetrachlorosalicylanilide, and tetracycline. Their effects on the aggregation of Abeta1-40, Abeta11-25, Abeta12-28, Abeta17-28, Abeta16-22, and Abeta16-22[methylated] analogues were characterized in terms of the integral widths and integrated intensities of the two characteristic reflections. Peptide Abeta11-25 with or without small molecules showed varying relative intensities but similar coherent lengths of 28-49 A in the intersheet and 171-221 A in the H-bonding directions. PTI-00703, however, abolished the H-bonding reflection. Among previously reported aromatic inhibitors for Abeta11-25, PTI-00703, tannic acid, and quinine were more effective than curcumin, morin, and melatonin based on the criterion of crystallite volume. For the N-methylated and control samples, there were no substantial differences in spacings and coherent lengths; however, the relative volumes of the beta-crystallites, which were calculated from the magnitude of the intensities, decreased with increase in concentration of Abeta16-22Me. This may be accounted for by the binding of Abeta16-22Me to the monomer or preamyloid oligomer of Abeta16-22. The fiber diffraction approach, which can help to specify whether an amyloidophilic compound acts by impeding hydrogen-bonding or by altering intersheet interactions, may help provide a rationale basis for the development of other therapeutic reagents.

Formation of amyloid fibrils in vitro by human gammaD-crystallin and its isolated domains.

PURPOSE: Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Previous studies have shown that bovine crystallins form amyloid fibers under denaturing conditions and amyloid fibers accumulate in the lens of mice carrying mutations in crystallin genes. Within differentiating lens fiber cells, crystallins may be exposed to low pH lysosome compartments. We have investigated whether human gammaD-crystallin forms amyloid fibrils in vitro, when exposed to low pH partially denaturing conditions. METHODS: Human gammaD-crystallin expressed and purified from E. coli, is stable and soluble at 37 degrees C, pH7, and refolds from the fully denatured state back to the native state under these conditions. Purified Human gammaD-crystallin as well as its isolated NH2- and COOH-terminal domains were incubated at acid pH and subsequently examined by transmission electron microscopy, absorption spectroscopy in the presence of Congo red, FTIR, and low-angle X-ray scattering. RESULTS: Incubation of the intact protein at 37 degrees C in 50 mM acetate buffer pH 3 at 50 mg/ml for 2 days, led to formation of a viscous, gel-like solution. Examination of negatively stained samples by transmission electron microscopy revealed linear, non-branching fibrils of variable lengths, with widths ranging from 15 to 35 nm. Incubation with the dye Congo red generated the spectral red shift associated with dye binding to amyloid. Low-angle X-ray scattering from samples showed clear meridional reflection at 4.7 A and a more diffuse reflection on the equator between 10 and 11 A which is the typical "cross-beta" X-ray fiber diffraction pattern for amyloid fibers. FTIR was used to follow the evolution of the secondary structure of gammaD-crystallin with time during incubation of the protein at pH 3. The native protein displayed a major band at 1640 cm-1 that converted during incubation at 37 degrees C to a band at 1616 cm-1. An additional band at 1689 cm-1 also appeared with time. The presence of bands in the regions about 1620 cm-1 and about 1680 cm-1 has been attributed to the formation of intermolecular beta-sheet structure that characterizes the fibrillar amyloid motif. The isolated NH2-terminal 1-82 and COOH-terminal 86-174 domains of HgammaD-crystallin also formed amyloid fibrils after incubation under the same conditions, but to a lesser extent than the full length. CONCLUSIONS: HgammaD-crystallin, as well as its isolated NH2-terminal 1-82 and COOH-terminal 86-174 domains of HgammaD-crystallin formed amyloid fibrils upon incubation at acid pH. Investigations of early stages in cataract formation within the lens will be required to assess whether amyloid fibrils play a role in the initiation of cataract in vivo.

Elucidation of residue-level structure and dynamics of polypeptides via isotope-edited infrared spectroscopy.

Infrared spectroscopy is a powerful tool for analyzing the structure of proteins and peptides. The amide I band is particularly sensitive to the strength and position of the hydrogen bonds that define secondary structure as well as dipole-dipole interactions that are affected by the geometry of the peptide backbone. The introduction of a single (13)C-labeled carbonyl into a peptide backbone results in a resolvable shoulder to the main amide I band, which can be analyzed as a separate peak. Thus, site-specific structural information can be obtained by sequential, systematic labeling of the backbone. This method of isotope-edited infrared spectroscopy is a tool for obtaining medium-resolution information about the backbone conformation and dynamics. This tool has been used to dissect the conformation and dynamics of alpha helices and amyloid aggregates, where the versatility of possible sampling with infrared spectroscopy is well-suited for studies of large-protein aggregates.

The alpha-helix folds more rapidly at the C-terminus than at the N-terminus.

The helix-coil dynamics of different sections of an alpha-helical model peptide were observed separately by nanosecond temperature jump experiments with IR detection on a series of isotopically labeled peptides. The results show that the helix-coil dynamics of the alpha-helical C-terminus are faster than those of the N-terminus.

Intersheet rearrangement of polypeptides during nucleation of {beta}-sheet aggregates.

Many neurodegenerative diseases are characterized by the accumulation of amyloid fibers in the brain, which can occur when a protein misfolds into an extended beta-sheet conformation. The nucleation of these beta-sheet aggregates is of particular interest, not only because it is the rate-determining step toward fiber formation but also because early, soluble aggregate species may be the cytotoxic entities in many diseases. In the case of the prion peptide H1 (residues 109-122 of the prion protein) stable amyloid fibers form only after the beta-strands of the peptide have adopted their equilibrium antiparallel beta-sheet configuration with residue 117 in register across all strands. In this article, we present the kinetic details of the realignment of these beta-strands from their fastformed nonequilibrium structure, which has no regular register of the strands, into the more ordered beta-sheets capable of aggregating into stable fibers. This process is likely the nucleating step toward the formation of stable fibers. Isotope-edited IR spectroscopy is used to monitor the alignment of the beta-strands by the introduction of a (13)C-labeled carbonyl at residue 117. Nonexponential kinetics is observed, with a complex dependence on concentration. The results are consistent with a mechanism in which the beta-sheet realigns by both the repeated detachment and annealing of strands in solution and reptation of polypeptide strands within an aggregate.

Experimental evidence for the reorganization of beta-strands within aggregates of the Abeta(16-22) peptide.

Amyloidogenic deposits that accumulate in brain tissue with the progression of Alzheimer's disease contain large amounts of the amyloid beta-peptide. A small fragment of this peptide, comprising residues 16-22 (Abeta(16-22)), forms beta-sheets in isolation, which then aggregate into amyloid fibrils. Here, using isotope edited infrared spectroscopy to probe the secondary structure of the peptide with residue level specificity, we are able to show conclusively that the beta-sheets formed are antiparallel and, following an anneal cycle or prolonged incubation, are in register with the central residue (Phe19) in alignment across all strands. The alignment of strands proceeds via a rapid interchange from one sheet to another. This realignment of the peptide strands into a more favorable registry may have important implications for therapeutics since previous work has shown that well aligned beta-sheets form more stable amyloid fibrils.

Correlations among morphology, beta-sheet stability, and molecular structure in prion peptide aggregates.

The misfolding of proteins into beta-sheets and the subsequent aggregation of these sheets into fibrous networks underlies many diseases. In this paper, the role of peptide structure in determining the ordering of beta-sheet aggregates and the morphology of fibrils and protofibrils is dissected. Using a series of peptides based on residues 109-122 of the Syrian hamster prion protein (H1) with a range of substitutions at position 117, the link between side chain interactions and beta-sheet thermal stability has been investigated. The thermal stability of beta-sheets is associated with the peptides' ability to adopt the same alignment as wild-type H1, with residue 117 in register across all beta-strands [Silva, R. A. G. D., Barber-Armstrong, W., and Decatur, S. M. (2003) J. Am. Chem. Soc. 125, 13674-13675]. These aligned strands are capable of forming long, rigid, and twisted fibrils (as visualized by atomic force microscopy) which are thermostable. Peptides which do not adopt this strand alignment aggregate to form thin, flexible, and smooth protofibrils. The ability to form ordered aggregates, and thus to form twisted fibrils, is modulated by the structure of the side chain of residue 117.

Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study.

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation. In this paper, we report the IR spectra as a function of TFE concentration for an alanine-rich peptide based on the repeat (AAKAA)(n)(). The IR spectra confirm that TFE desolvates the helical state of the peptide to a greater extent than the random coil state. Moreover, using a series of specifically (13)C-labeled peptides, the precise residues desolvated in the presence of TFE were identified. The residues most desolvated by TFE are the alanines located at position i - 4 in the sequence, where i is a lysine residue. This pattern of desolvation is consistent with molecular dynamics simulations which predict strong interactions between the lysine side chain at position n and the backbone carbonyl of the alanine at position i - 4. This is the first direct spectroscopic evidence of specific desolvation of helix backbone atoms in model alanine-rich peptides.

Empirical relationships between isotope-edited IR spectra and helix geometry in model peptides.

Infrared spectroscopy (IR) is commonly used to study secondary structure of both peptides and proteins. The amide I band is very sensitive to peptide secondary structure, and the conformation of a peptide can be probed at the residue level by introducing site-specific isotope-labels into the peptide backbone. The replacement of a carbonyl (12)C with a (13)C results in a approximately 40 cm(-1) shift in the amide I' band. The amide I bands of specifically labeled helices should vary systematically as a function of the number and relative spacing of the labeled residues; thus one should be able to describe the conformation of a polypeptide in substantial detail by probing the changes in IR spectra as a function of the number and positioning of isotope labels. In this study, we report IR spectra of a series of differently labeled helical peptides. A series of 25mer peptides were synthesized based on the repeat sequence (AAAAK)(n). We have varied the number and spacing of the labels on each peptide and studied the changes in the (12)C and (13)C amide I' band due to label position. Our results indicate that changing the number of labels changes the frequency and intensity of both the (12)C and the (13)C amide mode. We also found that varying the spacing between labels causes these amide peaks to shift. Isotope labeling, combined with IR spectroscopy and theoretical predictions, may generate a description of peptide backbone conformations at the residue level.

Nature of vibrational coupling in helical peptides: an isotopic labeling study.

Infrared (IR) and vibrational circular dichroism (VCD) spectra were measured for a series of isotopically ((13)C on two or more amide Cdouble bond]O) labeled, 25 residue, alpha-helical peptides of the sequence Ac-(AAAAK)(4)AAAAY-NH(2) that were also studied in the previous paper. Theoretical IR and VCD simulations were performed for correspondingly isotopically labeled Ac-A(24)-NHCH(3) constrained to an alpha-helical conformation by use of property tensor transfer from density functional theory (DFT) calculations on Ac-A(10)-NHCH(3). The simulations predicted and experiments confirmed that the vibrational coupling constants between i, i + 1 and i, i + 2 residues differ in sign, thus leading to a reversal of the (13)C VCD pattern and explaining the large shift in the (13)C amide I frequency as reported in the previous paper. The sign of the coupling constant remained consistent for larger label separation (with the exception of i, i + 4) and for more labels with uniform separation. Such effects confirm that the isotopically labeled group vibrations are essentially only coupled to each other and are effectively uncoupled from those of the unlabeled groups. This development confirms the utility of isotopic labels for site-specific structural studies with vibrational spectra. Observed spectral effects cannot be explained by considering only transition dipole coupling (TDC) between amide oscillators, particularly for smaller label separations, but the TDC and ab initio predicted couplings roughly converge at large separation.

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study.

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.

Probing the effect of side chains on the conformation and stability of helical peptides via isotope-edited infrared spectroscopy.

The mechanism of helix stabilization or destabilization by different amino acids has been the subject of several experimental and theoretical studies; these studies suggest that large or bulky side chains may modulate helix stability by altering the hydration of the helix backbone. In this paper, we report a spectroscopic study to determine the effect of alanine to leucine substitutions on the conformation and solvation of specific segments of a model helical peptide. A 25-residue, alanine-rich, helical peptide [Ac-(AAAAK)(4)-AAAAY-NH(2) (AKA)] and its two leucine variants [Ac-LLLLK-(AAAAK)(3)-AAAAY-NH(2) (LKA) and Ac-(AAAAK)(4)-LLLLY-NH(2) (AKL)] were characterized by infrared (IR) and electronic circular dichroism (ECD) spectroscopies. Introduction of (13)C isotopes into specific, consecutive, backbone carbonyls for certain blocks of each of the peptides mentioned above allows the IR spectra to be interpreted in terms of the conformation and solvation of specific residues within the helix. These isotope-edited IR spectra of the leucine peptides do not show evidence of a decrease in the degree of backbone solvation compared to the alanines, but suggest that the peptide may adopt a distorted conformation to accommodate the larger leucine side chains at the N-terminus. These experiments demonstrate the power of isotope-edited IR in dissecting subtle changes in helix conformation at the residue level.