Christopher P. Jaroniec

Atomic structure and hierarchical assembly of a cross-β amyloid fibril.

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril

The molecular conformation of peptide fragment 105–115 of transthyretin, TTR(105–115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105–115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105–114. Analysis of the 15N, 13CO, 13Cα, and 13Cβ chemical shifts allowed quantitative predictions to be made for the backbone torsion angles φ and ψ. Furthermore, four backbone 13C–15N distances were determined in two selectively 13C,15N-labeled fibril samples by using rotational-echo double-resonance NMR. The results show that TTR(105–115) adopts an extended β-strand conformation that is similar to that found in the native protein except for substantial differences in the vicinity of the proline residue.

De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy

The three-dimensional structure of the chemotactic peptide N-formyl-l-Met-l-Leu-l-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 13C–15N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly 13C,15N- and 15N-labeled samples. The peptides structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Amphiphilic Self‐Assembly of an n‐Type Nanotube

The electronic properties of p-conjugated materials depend on the nature of the interactions among the constituent chromophores. The p–p stacking interactions present in aggregated arrays of semiconductors provide pathways for charge transport and energy migration. Thus, the selfassembly of p-conjugated building blocks into discrete, onedimensional (1D) nanostructures is a powerful strategy to tune the properties of organic electronic materials. The majority of these approaches have produced twisted nanofibers of p-type chromophores. The exceptional electronic characteristics of carbon nanotubes have also inspired interest in versatile supramolecular approaches toward p-conjugated nanotubes. The availability of self-assembled organic nanotubes would provide greater modularity in their design and functionalization. However, examples of p-conjugated systems that assemble into well-defined nanotubes are relatively uncommon. Herein, we describe a 1D n-type nanotube formed by the bolaamphiphilic self-assembly of 1,4,5,8-naphthalenetetracarboxylic acid diimide (NDI) with l-lysine headgroups (Figure 1). We recently reported a simple method for fabricating n-type 1D nanostructures by the b-sheet assembly of dipeptide–NDI conjugates into either helical nanofibers or twisted nanoribbons. Time-resolved fluorescence anisotropy experiments showed enhanced energy migration within these nanostructures. Herein, we explore how the intermolecular electrostatic interactions derived from the lysine headgroups in bolaamphiphile A (Figure 1 a), in conjunction with p–p association among the NDI chromophores, drive the self-assembly process in water toward soluble, well-ordered 1D nanotubes. Bolaamphiphile A was constructed by imidation of 1,4,5,8-naphthalenetetracarboxylic acid dianhydride with two equivalents of Boc-l-lysine, followed by TFA deprotection (Supporting Information, Scheme S1). Bolaamphiphile A formed a transparent gel in water at concentrations as low as 1% (w/w) (1.9 mm ; Figure 1b, red inset), and was stable in the gel state for several months. Transmission electron microscopy (TEM) of a negatively stained sample of A revealed the formation of micrometer-long nanotubes with uniform diameters of (12 1) nm (Figure 1b). The nanotubes appeared as two white, parallel lines separated by a dark center, which is consistent with the cross-sectional view of a hollow tubular structure filled with the negative stain, uranyl acetate (Figure 1 b). The thickness of the wall was approximately (2.5 0.5) nm. A few nanorings, albeit rare, could also be observed in the TEM images (red arrows in Figure 1 b), with external diameters of 12 nm and wall thicknesses of 2.5 nm, which are identical with the nanotube dimensions. Tapping-mode AFM imaging of dilute bolaamphiphile A gel samples (250 mm) on mica also revealed high-aspect ratio assemblies with cross-sectional heights of about 9 nm, which were slightly smaller than those observed by TEM; this effect Figure 1. a) Structures of lysine-based bolaamphiphiles A (R =O ) and B (R = OMe) and the assembly of A into rings, which stack to give tubes. The blue sections of A undergo hydrophobic p–p stacking interactions, and the red sections electrostatic interactions. b) TEM image of bolaamphiphile A in water (250 mm ; carbon-coated copper grid); 2% (w/w) uranyl acetate as negative stain. Blue insets: Two nanotubes and one nanoring. c) Tapping-mode AFM image of bolaamphiphile A in water (250 mm) on freshly cleaved mica. Red inset: Section analysis showing uniform height of the assemblies. Height indicated by red arrows: ca. 9 nm.

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 13C,15N-labeled huPrP23–144. Surprisingly, we find that signals from ≈100 residues (i.e., ≈80% of the sequence) are not detected above approximately −20°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 13C and 15N linewidths of ≈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 13C secondary chemical shifts identified likely β-strand segments within this core region, including β-strand 130–139 containing critical residues 138 and 139. In contrast to this relatively rigid, β-sheet-rich amyloid core, the remaining residues in huPrP23–144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.

Conformational flexibility of Y145stop human prion protein amyloid fibrils probed by solid-state nuclear magnetic resonance spectroscopy

Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

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.

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 nuclear magnetic resonance. However, the paucity of >5 Å distance restraints, 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 analogs 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-Cu2+ mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone 15N PREs corresponding to ~10-20 Å distances 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.

Insights into the Mode of Action of a Putative Zinc Transporter CzrB in Thermus thermophilus

The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.

Nmrglue: an open source Python package for the analysis of multidimensional NMR data

Nmrglue, an open source Python package for working with multidimensional NMR data, is described. When used in combination with other Python scientific libraries, nmrglue provides a highly flexible and robust environment for spectral processing, analysis and visualization and includes a number of common utilities such as linear prediction, peak picking and lineshape fitting. The package also enables existing NMR software programs to be readily tied together, currently facilitating the reading, writing and conversion of data stored in Bruker, Agilent/Varian, NMRPipe, Sparky, SIMPSON, and Rowland NMR Toolkit file formats. In addition to standard applications, the versatility offered by nmrglue makes the package particularly suitable for tasks that include manipulating raw spectrometer data files, automated quantitative analysis of multidimensional NMR spectra with irregular lineshapes such as those frequently encountered in the context of biomacromolecular solid-state NMR, and rapid implementation and development of unconventional data processing methods such as covariance NMR and other non-Fourier approaches. Detailed documentation, install files and source code for nmrglue are freely available at http://nmrglue.com. The source code can be redistributed and modified under the New BSD license.