Protein-solvent interfaces in human Y145Stop prion protein amyloid fibrils probed by paramagnetic solid-state NMR spectroscopy.

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

Structural Studies of Amyloid Fibrils by Paramagnetic Solid-State Nuclear Magnetic Resonance Spectroscopy.

Application of paramagnetic solid-state NMR to amyloids is demonstrated, using Y145Stop human prion protein modified with nitroxide spin-label or EDTA-Cu2+ tags as a model. By using sample preparation protocols based on seeding with preformed fibrils, we show that paramagnetic protein analogs can be induced into adopting the wild-type amyloid structure. Measurements of residue-specific intramolecular and intermolecular paramagnetic relaxation enhancements enable determination of protein fold within the fibril core and protofilament assembly. These methods are expected to be widely applicable to other amyloids and protein assemblies.

Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag.

Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu(2+) binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC-Cu(2+) K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu(2+) center experience considerable transverse PREs leading to severely attenuated resonances in 2D (15)N-(13)C correlation spectra. For more distant residues, electron-nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for (15)N-Cu(2+) distances up to ~20 Å.

Protein structure determination with paramagnetic solid-state NMR spectroscopy.

Many structures of the proteins and protein assemblies that play central roles in fundamental biological processes and disease pathogenesis are not readily accessible via the conventional techniques of single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR). On the other hand, many of these challenging biological systems are suitable targets for atomic-level structural and dynamic analysis by magic-angle spinning (MAS) solid-state NMR spectroscopy, a technique that has far less stringent limitations on the molecular size and crystalline state. Over the past decade, major advances in instrumentation and methodology have prompted rapid growth in the field of biological solid-state NMR. However, despite this progress, one challenge for the elucidation of three-dimensional (3D) protein structures via conventional MAS NMR methods is the relative lack of long-distance data. Specifically, extracting unambiguous interatomic distance restraints larger than ∼5 Šfrom through-space magnetic dipole-dipole couplings among the protein (1)H, (13)C, and (15)N nuclei has proven to be a considerable challenge for researchers. It is possible to circumvent this problem by extending the structural studies to include several analogs of the protein of interest, intentionally modified to contain covalently attached paramagnetic tags at selected sites. In these paramagnetic proteins, the hyperfine couplings between the nuclei and unpaired electrons can manifest themselves in NMR spectra in the form of relaxation enhancements of the nuclear spins that depend on the electron-nucleus distance. These effects can be significant for nuclei located up to ∼20 Šaway from the paramagnetic center. In this Account, we discuss MAS NMR structural studies of nitroxide and EDTA-Cu(2+) labeled variants of a model 56 amino acid globular protein, B1 immunoglobulin-binding domain of protein G (GB1), in the microcrystalline solid phase. We used a set of six EDTA-Cu(2+)-tagged GB1 mutants to rapidly determine the global protein fold in a de novo fashion. Remarkably, these studies required quantitative measurements of only approximately four or five backbone amide (15)N longitudinal paramagnetic relaxation enhancements per residue, in the complete absence of the usual internuclear distance restraints. Importantly, this paramagnetic solid-state NMR methodology is general and can be directly applied to larger proteins and protein complexes for which a significant fraction of the signals can be assigned in standard 2D and 3D MAS NMR chemical shift correlation spectra.

Histone H3 and H4 N-terminal tails in nucleosome arrays at cellular concentrations probed by magic angle spinning NMR spectroscopy.

Chromatin is a supramolecular assembly of DNA and histone proteins, organized into nucleosome repeat units. The dynamics of chromatin organization regulates DNA accessibility to eukaryotic transcription and DNA repair complexes. Yet, the structural and dynamic properties of chromatin at high concentrations characteristic of the cellular environment (>∼200 mg/mL) are largely unexplored at the molecular level. Here, we apply MAS NMR to directly probe the dynamic histone protein regions in (13)C,(15)N-enriched recombinant nucleosome arrays at cellular chromatin concentrations and conditions designed to emulate distinct states of DNA condensation, with focus on the flexible H3 and H4 N-terminal tails which mediate chromatin compaction. 2D (1)H-(13)C and (1)H-(15)N spectra reveal numerous correlations for H3 and H4 backbone and side-chain atoms, enabling identification of specific residues making up the dynamically disordered N-terminal tail domains. Remarkably, we find that both the H3 and H4 N-terminal tails are overall dynamic even in a highly condensed state. This significant conformational flexibility of the histone tails suggests that they remain available for protein binding in compact chromatin states to enable regulation of heterochromatin. Furthermore, our study provides a foundation for quantitative structural and dynamic investigations of chromatin at physiological concentrations.

Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils.

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the ß-sheet core of the PrP23-144 amyloid is composed of two ß-strand regions encompassing residues ∼113-125 and ∼130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered ß-core extended N-terminally to residue ∼106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal ß-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.

Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo.

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of 15N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR.

Magic-angle spinning solid-state NMR measurements of (15)N longitudinal paramagnetic relaxation enhancements (PREs) in (13)C,(15)N-labeled proteins modified with Cu(2+)-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108-8120, 2009). Using the EDTA-Cu(2+) K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular (15)N-Cu(2+) interactions we have determined a nearly complete set of longitudinal (15)N PREs for a series of microcrystalline samples containing ~10, 15 and 25 mol percent of the (13)C,(15)N-labeled EDTA-Cu(2+)-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental (15)N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu(2+) tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu(2+) ions. To probe this issue we performed a Cu(2+) titration study for K28C-EDTA GB1 monitored by 2D (15)N-(1)H solution-state NMR, which revealed that while for Cu(2+):protein molar ratios of ≤ 1.0 Cu(2+) binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu(2+) ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu(2+) binding sites, and indeed many of these residues were ones found to display the elevated longitudinal (15)N PREs in the solid phase.

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils.

A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by (13)C,(15)N-labeled huPrP23-144. Surprisingly, we find that signals from approximately 100 residues (i.e., approximately 80% of the sequence) are not detected above approximately -20 degrees C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112-141. These resonances are remarkably narrow, exhibiting average (13)C and (15)N linewidths of approximately 0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23-144 amyloid encompassing residues 112-141. Analysis of (13)C secondary chemical shifts identified likely beta-strand segments within this core region, including beta-strand 130-139 containing critical residues 138 and 139. In contrast to this relatively rigid, beta-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.

Rapid acquisition of multidimensional solid-state NMR spectra of proteins facilitated by covalently bound paramagnetic tags.

We describe a condensed data collection approach that facilitates rapid acquisition of multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) spectra of proteins by combining rapid sample spinning, optimized low-power radio frequency pulse schemes and covalently attached paramagnetic tags to enhance protein (1)H spin-lattice relaxation. Using EDTA-Cu(2+)-modified K28C and N8C mutants of the B1 immunoglobulin binding domain of protein G as models, we demonstrate that high resolution and sensitivity 2D and 3D SSNMR chemical shift correlation spectra can be recorded in as little as several minutes and several hours, respectively, for samples containing approximately 0.1-0.2 micromol of (13)C,(15)N- or (2)H,(13)C,(15)N-labeled protein. This mode of data acquisition is naturally suited toward the structural SSNMR studies of paramagnetic proteins, for which the typical (1)H longitudinal relaxation time constants are inherently a factor of at least approximately 3-4 lower relative to their diamagnetic counterparts. To illustrate this, we demonstrate the rapid site-specific determination of backbone amide (15)N longitudinal paramagnetic relaxation enhancements using a pseudo-3D SSNMR experiment based on (15)N-(13)C correlation spectroscopy, and we show that such measurements yield valuable long-range (15)N-Cu(2+) distance restraints which report on the three-dimensional protein fold.

Expression and purification of a recombinant amyloidogenic peptide from transthyretin for solid-state NMR spectroscopy.

We describe the expression and purification of a model amyloidogenic peptide comprising residues 105-115 of human transthyretin (TTR105-115). Recombinant TTR105-115, which does not contain any non-native residues, was prepared as part of a fusion protein construct with a highly soluble B1 immunoglobulin binding domain of protein G (GB1), with typical yields of approximately 4 mg/L of uniformly (13)C,(15)N-enriched HPLC-purified peptide per liter of minimal media culture. Amyloid fibrils formed by recombinant TTR105-115 were characterized by transmission electron microscopy and solid-state NMR spectroscopy, and found to be comparable to synthetic TTR105-115 fibrils. These results establish recombinant TTR105-115 as a valuable model system for the development of new solid-state NMR techniques for the atomic-level characterization of amyloid architecture.

Paramagnetic ions enable tuning of nuclear relaxation rates and provide long-range structural restraints in solid-state NMR of proteins.

Magic-angle-spinning solid-state nuclear magnetic resonance (SSNMR) studies of natively diamagnetic uniformly (13)C,(15)N-enriched proteins, intentionally modified with side chains containing paramagnetic ions, are presented, with the aim of using the concomitant nuclear paramagnetic relaxation enhancements (PREs) as a source of long-range structural information. The paramagnetic ions are incorporated at selected sites in the protein as EDTA-metal complexes by introducing a solvent-exposed cysteine residue using site-directed mutagenesis, followed by modification with a thiol-specific reagent, N-[S-(2-pyridylthio)cysteaminyl]EDTA-metal. Here, this approach is demonstrated for the K28C and T53C mutants of B1 immunoglobulin-binding domain of protein G (GB1), modified with EDTA-Mn(2+) and EDTA-Cu(2+) side chains. It is shown that incorporation of paramagnetic moieties, exhibiting different relaxation times and spin quantum numbers, facilitates the convenient modulation of longitudinal (R(1)) and transverse (R(2), R(1rho)) relaxation rates of the protein (1)H, (13)C, and (15)N nuclei. Specifically, the EDTA-Mn(2+) side chain generates large distance-dependent transverse relaxation enhancements, analogous to those observed previously in the presence of nitroxide spin labels, while this phenomenon is significantly attenuated for the Cu(2+) center. Both Mn(2+) and Cu(2+) ions cause considerable longitudinal nuclear PREs. The combination of negligible transverse and substantial longitudinal relaxation enhancements obtained with the EDTA-Cu(2+) side chain is especially advantageous, because it enables structural restraints for most sites in the protein to be readily accessed via quantitative, site-resolved measurements of nuclear R(1) rate constants by multidimensional SSNMR methods. This is demonstrated here for backbone amide (15)N nuclei, using methods based on 2D (15)N-(13)C chemical shift correlation spectroscopy. The measured longitudinal PREs are found to be highly correlated with the proximity of the Cu(2+) ion to (15)N spins, with significant effects observed for nuclei up to approximately 20 A away, thereby providing important information about protein structure on length scales that are inaccessible to traditional SSNMR techniques.

Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy.

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ∼10-20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10-50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.

13C and 15N chemical shift assignments of mammalian Y145Stop prion protein amyloid fibrils.

The Y145Stop prion protein (PrP23-144), which has been linked to the development of a heritable prionopathy in humans, is a valuable in vitro model for elucidating the structural and molecular basis of amyloid seeding specificities. Here we report the sequential backbone and side-chain 13C and 15N assignments of mouse and Syrian hamster PrP23-144 amyloid fibrils determined by using 2D and 3D magic-angle spinning solid-state NMR. The assigned chemical shifts were used to predict the secondary structures for the core regions of the mouse and Syrian hamster PrP23-144 amyloids, and the results compared to those for human PrP23-144 amyloid, which has previously been analyzed by solid-state NMR techniques.

Species-dependent structural polymorphism of Y145Stop prion protein amyloid revealed by solid-state NMR spectroscopy.

One of the most puzzling aspects of the prion diseases is the intricate relationship between prion strains and interspecies transmissibility barriers. Previously we have shown that certain fundamental aspects of mammalian prion propagation, including the strain phenomenon and species barriers, can be reproduced in vitro in seeded fibrillization of the Y145Stop prion protein variant. Here, we use solid-state nuclear magnetic resonance spectroscopy to gain atomic level insight into the structural differences between Y145Stop prion protein amyloids from three species: human, mouse, and Syrian hamster. Remarkably, we find that these structural differences are largely controlled by only two amino acids at positions 112 and 139, and that the same residues appear to be key to the emergence of structurally distinct amyloid strains within the same protein sequence. The role of these residues as conformational switches can be rationalized based on a model for human Y145Stop prion protein amyloid, providing a foundation for understanding cross-seeding specificity.Prion diseases can be transmitted across species. Here the authors use solid-state NMR to study prion protein (PrP) amyloids from human, mouse and Syrian hamster and show that their structural differences are mainly governed by two residues, which helps to understand interspecies PrP propagation on a molecular level.

Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy.

Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state NMR spectroscopy. However, the paucity of distance restraints >5 Å, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here, we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogues of the protein of interest containing covalently attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu(2+) mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone (15)N PREs corresponding to distances of ~10-20 Å were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.

Determination of methyl 13C-15N dipolar couplings in peptides and proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy.

We describe three- and four-dimensional semiconstant-time transferred echo double resonance (SCT-TEDOR) magic-angle spinning solid-state nuclear magnetic resonance (NMR) experiments for the simultaneous measurement of multiple long-range (15)N-(13)C(methyl) dipolar couplings in uniformly (13)C, (15)N-enriched peptides and proteins with high resolution and sensitivity. The methods take advantage of (13)C spin topologies characteristic of the side-chain methyl groups in amino acids alanine, isoleucine, leucine, methionine, threonine, and valine to encode up to three distinct frequencies ((15)N-(13)C(methyl) dipolar coupling, (15)N chemical shift, and (13)C(methyl) chemical shift) within a single SCT evolution period of initial duration approximately 1(1)J(CC) (where (1)J(CC) approximately 35 Hz, is the one-bond (13)C(methyl)-(13)C J-coupling) while concurrently suppressing the modulation of NMR coherences due to (13)C-(13)C and (15)N-(13)C J-couplings and transverse relaxation. The SCT-TEDOR schemes offer several important advantages over previous methods of this type. First, significant (approximately twofold to threefold) gains in experimental sensitivity can be realized for weak (15)N-(13)C(methyl) dipolar couplings (corresponding to structurally interesting, approximately 3.5 A or longer, distances) and typical (13)C(methyl) transverse relaxation rates. Second, the entire SCT evolution period can be used for (13)C(methyl) and/or (15)N frequency encoding, leading to increased spectral resolution with minimal additional coherence decay. Third, the experiments are inherently "methyl selective," which results in simplified NMR spectra and obviates the use of frequency-selective pulses or other spectral filtering techniques. Finally, the (15)N-(13)C cross-peak buildup trajectories are purely dipolar in nature (i.e., not influenced by J-couplings or relaxation), which enables the straightforward extraction of (15)N-(13)C(methyl) distances using an analytical model. The SCT-TEDOR experiments are demonstrated on a uniformly (13)C, (15)N-labeled peptide, N-acetyl-valine, and a 56 amino acid protein, B1 immunoglobulin-binding domain of protein G (GB1), where the measured (15)N-(13)C(methyl) dipolar couplings provide site-specific information about side-chain dihedral angles and the packing of protein molecules in the crystal lattice.

13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy.

Complete 13C and 15N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state shifts.