Amyloid fibril structure of peptides and proteins by magic angle spinning NMR spectroscopy and dynamic nuclear polarization

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.Vita. Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.Amyloid fibrils are insoluble, non-crystalline protein filaments associated with a number of diseases such as Alzheimer's and Type Il diabetes. They can have a functional role in different organisms and many proteins and peptides have been found to form amyloid fibrils in vitro. We have used magic angle spinning (MAS) NMR spectroscopy to investigate the structure of two amyloid fibril systems - an 11- residue segment from the disease-related protein transthyretin (TTR); and P2- microglobulin (32m), a 99-residue protein associated with dialysis-related amyloidosis. The TTR(105-115) case exemplifies our efforts to characterize the hierarchy of structures present in the fibril form, including the organization of the Pstrands into P-sheets (tertiary structure), the P-sheet interface that defines each protofilament (quaternary structure), and the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts were guided by information obtained from other methods such as cryo-electron microscopy and atomic force microscopy, and resulted in the very first atomic resolution structure of a complete amyloid fibril. We have extended the methods used in the TTR(105-115) structure determination procedure to the fibrils formed by 2m, a process complicated not only by the much larger size of the protein involved but also by the high degree of dynamics exhibited in these fibrils. Nevertheless, we were able to characterize the secondary structure of the protein in the fibril form, and the tertiary and quaternary interactions within the fibrils. In addition, we have compared at the molecular level @2m fibrils formed under different conditions, in an effort to characterize the origins of fibril polymorphism for this protein sequence. Our work on amyloid fibrils has also benefited extensively from the development of dynamic nuclear polarization, a method used to enhance the sensitivity of MAS NMR experiments, leading to unprecedented gains in signal-to-noise ratios and acquisition times.by Galia Tzvetanova Debelouchina.Ph.D

Secondary structure in the core of amyloid fibrils formed from human βm and its truncated variant Δn6

Amyloid fibrils formed from initially soluble proteins with diverse sequences are associated with an array of human diseases. In the human disorder, dialysis-related amyloidosis (DRA), fibrils contain two major constituents, full-length human β-microglobulin (hβm) and a truncation variant, ΔN6 which lacks the N-terminal six amino acids. These fibrils are assembled from initially natively folded proteins with an all antiparallel β-stranded structure. Here, backbone conformations of wild-type hβm and ΔN6 in their amyloid forms have been determined using a combination of dilute isotopic labeling strategies and multidimensional magic angle spinning (MAS) NMR techniques at high magnetic fields, providing valuable structural information at the atomic-level about the fibril architecture. The secondary structures of both fibril types, determined by the assignment of ∼80% of the backbone resonances of these 100- and 94-residue proteins, respectively, reveal substantial backbone rearrangement compared with the location of β-strands in their native immunoglobulin folds. The identification of seven β-strands in hβm fibrils indicates that approximately 70 residues are in a β-strand conformation in the fibril core. By contrast, nine β-strands comprise the fibrils formed from ΔN6, indicating a more extensive core. The precise location and length of β-strands in the two fibril forms also differ. The results indicate fibrils of ΔN6 and hβm have an extensive core architecture involving the majority of residues in the polypeptide sequence. The common elements of the backbone structure of the two proteins likely facilitates their ability to copolymerize during amyloid fibril assembly

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a â-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C- and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. Keyword: [PSI+] prion; solid-state NMR; amyloid; Sup35; dynamic nuclear polarizationNational Institutes of Health (U.S.) (Grants GM-025874)National Institutes of Health (U.S.) (Grants EB-003151)National Institutes of Health (U.S.) (Grants EB-002804)National Institutes of Health (U.S.) (Grants EB-002026

Dynamic Nuclear Polarization-Enhanced Solid-State NMR Spectroscopy of GNNQQNY Nanocrystals and Amyloid Fibrils

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026

Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular [superscript 15]N–[superscript 13]C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.National Institutes of Health (U.S.) (Grant EB-003151)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and [alpha]-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a [beta]-sheet arrangement reminiscent of the [beta]-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Solid-State Dynamic Nuclear Polarization at 263 GHz: Spectrometer Design and Experimental Results

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz 1H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water–glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.National Institutes of Health (U.S.) (NIH grant EB-002804)National Institutes of Health (U.S.) (NIH grant EB-002026

Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

This work was funded by Wellcome Senior Fellowship 095062, Wellcome Trust grants 094090, 099149 and 097945. ALH was funded by an EMBO long term fellowship ALTF 380–2015 co-funded by the European Commission (LTFCOFUND2013, GA-2013–609409).ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein-DNA interactions. Here, we present structures of the Saccharomyces cerevisiae Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins. The structure provides connectivity between rearrangement of ATPase lobes to a closed, nucleotide bound state and the sensing of linker DNA. Two turns of linker DNA are prised off the surface of the histone octamer as a result of Chd1 binding, and both the histone H3 tail and ubiquitin conjugated to lysine 120 are re-orientated towards the unravelled DNA. This indicates how changes to nucleosome structure can alter the way in which histone epitopes are presented.Publisher PDFPeer reviewe