Glutamine Side Chain 13C?18O as a Nonperturbative IR Probe of Amyloid Fibril Hydration and Assembly.
ABSTRACT: Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain 13C?18O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain 13C?18O. We use side chain 13C?18O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the 13C?18O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain 13C?18O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies.
Project description:Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state nuclear magnetic resonance (NMR) structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C labeling schemes to investigate structural features of ?-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the 13C-13C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of nativelike CBEF and DAGH ?-sheet structures, and the mutant TTR amyloids adopt a similar amyloid core structure with nativelike CBEF and AGH ?-structures. However, the V30M mutant amyloid was shown to have a different DA ?-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA ?-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of the methionine side chain also revealed that the side chain of residue Met-30 pointing between strands D and A is not protected from oxidation in the V30M mutant, while protected in the native state, supporting the possibility that the DA ?-structure might be disrupted in the V30M mutant amyloid.
Project description:The peptide self-assembly mimic (PSAM) from the outer surface protein A (OspA) can form highly stable but soluble beta-rich self-assembly-like structures similar to those formed by native amyloid-forming peptides. However, unlike amyloids that predominantly form insoluble aggregates, PSAMs are highly water-soluble. Here, we characterize the conformations of these soluble beta-sheet-rich assemblies. We simulate PSAMs with different-sized beta-sheets in the presence and absence of end-capping proteins using all-atom explicit-solvent molecular dynamics, comparing the structural stability, conformational dynamics, and association force. Structural and free-energy comparisons among beta-sheets with different numbers of layers and sequences indicate that in similarity to amyloids, the intersheet side chain-side chain interactions and hydrogen bonds combined with intrasheet salt bridges are the major driving forces in stabilizing the overall structural organization. A detailed structural analysis shows that in similarity to amyloid fibrils, all wild-type and mutated PSAM structures display twisted and bent beta-sheets to some extent, implying that a twisted and bent beta-sheet is a general motif of beta-rich assemblies. Thus, our studies indicate that soluble beta-sheet-rich peptide self-assemblies can provide good amyloid mimics, and as such confirm on the atomic scale that they are excellent systems for amyloid studies. These results provide further insight into the usefulness of such mimics for nanostructure design.
Project description:The formation and deposition of amyloids is associated with many diseases. ?-Sheet secondary structure is a common feature of amyloids, but the packing of sheets against one another is distinctive relative to soluble proteins. Standard methods that rely on perturbing a polypeptide's sequence and evaluating impact on folding can be problematic for amyloid aggregates because a single sequence can adopt multiple conformations and diverse packing arrangements. We describe initial steps toward a minimum-sized, soluble model system for the amyloid state that supports comparisons among sequence variants. Critical to this goal is development of a new linking strategy to enable intersheet association mediated by side chain interactions, which is characteristic of the amyloid state. The linker design we identified should ultimately support exploration of relationships between sequence and amyloid state stability for specific strand-association modes.
Project description:The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of ?-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed ?/? structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C?(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the ?-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.
Project description:Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single 13C?18O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, 13C?18O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the 13C?18O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13C?18O labels at different positions in HP36 show drastic differences in the midpoint of their transitions ( Tm), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for 13C?18O A57 is 2.4e + 05 s-1 whereas for 13C?18O L69 HP36 the relaxation rate is 5.1e + 05 s-1, two times faster. The observation of site-dependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.
Project description:The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel ?-sheet amyloids including Tau and ?-synuclein (?Syn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and ?Syn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel ?-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of ?Syn amyloid, which suggest significance of compatibility between ?-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of ?Syn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.
Project description:Amyloids are highly organized cross-beta-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function, as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. We found that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross-beta-sheet-rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.
Project description:The 37-residue human islet amyloid polypeptide (hIAPP or amylin) self-assembles into fibers, the assembly of which has been associated with the disease mechanism of type II diabetes. Infrared spectroscopy in conjunction with isotope labeling is proving to be a powerful tool for studying the aggregation process of hIAPP and other amyloid forming proteins with residue specific structure and kinetic information, but the relationship between the spectroscopic observables and the structure is not fully established. We report a detailed analysis of the linear and 2D IR spectra of hIAPP fibers isotope labeled at seven different residue positions. The features of the 2D IR spectra, including the frequencies, linewidths, intensities, and polarization dependence of the diagonal and cross-peaks, rely heavily on the position of the isotope labeled residue. In order to understand how these measured parameters depend on fiber secondary and tertiary structure, we have simulated 1D and 2D IR spectra utilizing idealized structural models in addition to a recently published solid-state NMR based model of the amyloid fibril. The analysis provides a more rigorous foundation for interpreting the infrared spectra of amyloids. In addition, we demonstrate that 2D IR spectra can be employed to distinguish between residues in beta-sheets versus those in turn regions, and that transitional residues between secondary structures can be identified by the suppression of their cross-peaks in 2D IR spectra. This latter approach is not limited to amyloid fibrils and will be generally useful in identifying regions of secondary structure in proteins using 2D IR spectroscopy and isotope labeling.
Project description:An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of ?-helix and a gain of ?-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel ?-sheets instead of the more common parallel ?-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel ?-sheet-rich amyloids can be functional forms.
Project description:We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the ?-amyloid (A?1-40 and A?1-42) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm-1 in A? fibrils that does not appear in other A? aggregates, even those with predominantly ?-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm-1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm-1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated A? samples deplete the 1625 cm-1 band but do not affect the 1610 cm-1 band, demonstrating that the 1610 cm-1 band is due to very stable fibrils. We demonstrate that the 1610 cm-1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on A? crystal structures. It was not previously possible to distinguish stable A? fibrils from the less stable ?-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on A? aggregation, fibril formation, and toxicity.