David A. Middleton

Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20−29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.

Inhibitors of protein aggregation and toxicity

The aggregation of numerous peptides or proteins has been linked to the onset of disease, including Abeta (amyloid beta-peptide) in AD (Alzheimers disease), asyn (alpha-synuclein) in Parkinsons disease and amylin in Type 2 diabetes. Diverse amyloidogenic proteins can often be cut down to an SRE (self-recognition element) of as few as five residues that retains the ability to aggregate. SREs can be used as a starting point for aggregation inhibitors. In particular, N-methylated SREs can bind to a target on one side, but have hydrogen-bonding blocked on their methylated face, interfering with further assembly. We applied this strategy to develop Abeta toxicity inhibitors. Our compounds, and a range of compounds from the literature, were compared under the same conditions, using biophysical and toxicity assays. Two N-methylated D-peptide inhibitors with unnatural side chains were the most effective and can reverse Abeta-induced inhibition of LTP (long-term potentiation) at concentrations as low as 10 nM. An SRE in asyn (VAQKTV) was identified using solid-state NMR. When VAQKTV was N-methylated, it was able to disrupt asyn aggregation. N-methylated derivatives of the SRE of amylin are also able to inhibit amylin aggregation.

Membrane protein structure: the contribution and potential of novel solid state NMR approaches.

Alternative methods for describing molecular detail for large integral membrane proteins are required in the absence of routine crystallographic approaches. Novel solid state NMR methods, devised for the study of large molecular assemblies, are now finding applications in biological systems, including integral membrane proteins. Wild-type and genetically engineered proteins can be investigated and detailed information about side chains, prosthetic groups, ligands (e.g. drugs) and binding sites can be deduced. The molecular structure and dynamics of selected parts of the proteins are accessible by a range of different solid state NMR approaches. Inter- and intra-atomic distances can be determined rather accurately (within ångströms) and the orientation of molecular bonds (within 2 degrees) can be measured in ideal cases. Here, a brief description of the methods is given and then some specific examples described with an indication of the future potential for the approaches in studying membrane proteins. It is anticipated that this emerging NMR methodology will be more widely used in the future, not only for resolving local structure, but also for more expansive descriptions of membrane protein structure at atomic resolution.

Design of an N-Methylated Peptide Inhibitor of α-Synuclein Aggregation Guided by Solid-State NMR§

Many neurodegenerative diseases are associated with the aggregation of misfolded proteins into amyloid oligomers or fibrils that are deposited as pathological lesions within areas of the brain. An attractive therapeutic strategy for preventing or ameliorating amyloid formation is to identify agents that inhibit the onset or propagation of protein aggregation. Here we demonstrate how solid-state nuclear magnetic resonance (ssNMR) may be used to identify key residues within amyloidogenic protein sequences that may be targeted to inhibit the aggregation of the host protein. For alpha-synuclein, the major protein component of Lewy bodies associated with Parkinsons disease, we have used a combination of ssNMR and biochemical data to identify the key region for self-aggregation of the protein as residues 77-82 (VAQKTV). We used our new structural information to design a peptide derived from residues 77 to 82 of alpha-synuclein with an N-methyl group at the C-terminal residue, which was able to disrupt the aggregation of alpha-synuclein. Thus, we have shown how structural data obtained from ssNMR can guide the design of modified peptides for use as amyloid inhibitors, as a primary step toward developing therapeutic compounds for prevention and/or treatment of amyloid diseases.

Amyloid-derived peptide forms self-assembled monolayers on gold nanoparticle with a curvature-dependent β-sheet structure.

Using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (SSNMR) techniques, the secondary structure of peptides anchored on gold nanoparticles of different sizes is investigated. The structure of the well-studied CALNN-capped nanoparticles is compared to the structure of nanoparticles capped with a new cysteine-terminated peptide, CFGAILSS. The design of that peptide is derived from the minimal amyloidogenic sequence FGAIL of the human islet polypeptide amylin. We demonstrate that CFGAILSS forms extended fibrils in solution. When constrained at a nanoparticle surface, CFGAILSS adopts a secondary structure markedly different from CALNN. Taking into account the surface selection rules, the FTIR spectra of CFGAILSS-capped gold nanoparticles indicate the formation of β-sheets which are more prominent for 25 nm diameter nanoparticles than for 5 nm nanoparticles. No intermolecular (13)C-(13)C dipolar coupling is detected with rotational resonance SSNMR for CALNN-capped nanoparticles, while CALNN is in a random coil configuration. Coupling is detected for CFGAILSS-capped gold nanoparticles, however, consistent with an intermolecular (13)C-(13)C distance of 5.0 ± 0.3 Å, in agreement with intermolecular hydrogen bonding in a parallel β-sheet structure.

A Model of Reversible Inhibitors in the Gastric H+/K+-ATPase Binding Site Determined by Rotational Echo Double Resonance NMR

Several close analogues of the noncovalent H+/K+-ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC50 values for porcine H+/K+-ATPase inhibition falling in the sub-10 μm range. Deuterium NMR spectra of a2H-labeled inhibitor titrated into H+/K+-ATPase membranes revealed that 80–100% of inhibitor was bound to the protein, and K+-competition2H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from13C–19F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H+/K+-ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe126 close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate.

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.

Insights into the Molecular Architecture of a Peptide Nanotube Using FTIR and Solid‐State NMR Spectroscopic Measurements on an Aligned Sample

Queuing up: Molecular orientation within macroscopically aligned nanotubes of the peptide AAAAAAK can be studied by solid-state NMR and IR spectroscopy. Line shape analysis of the NMR spectra indicates that the peptide N-H bonds are tilted 65-70° relative to the nanotube long axis. Re-evaluation of earlier X-ray fiber diffraction data suggests that the peptide molecules are hydrogen-bonded in a helical arrangement along the nanotube axis.

The effects of alpha-synuclein on phospholipid vesicle integrity: a study using 31P NMR and electron microscopy

Associations between the 140 amino acid protein α-synuclein (asyn) and presynaptic vesicles may play a role in maintaining synaptic plasticity and neurotransmitter release. These physiological processes may involve disruption and fusion of vesicles, arising from interactions between specific regions of asyn, including the highly basic N-terminal domain, and the surface of vesicles. This work investigates whether asyn affects the integrity of model unilamellar vesicles of varying size and phospholipid composition, by monitoring paramagnetic Mn2+-induced broadening of peaks in the 31P nuclear magnetic resonance spectrum of the lipid head groups. It is shown that asyn increases the permeability to Mn2+ of both large (200nm diameter) and small (50nm diameter) vesicles composed of zwitterionic phosphatidylcholine and anionic phosphatidylglycerol at protein/lipid molar ratios as low as 1:2000. Further experiments on peptides corresponding to sequences in the N-terminal (10–48), C-terminal (120–140) and central hydrophobic (71–82) regions of asyn suggest that single regions of the protein are capable of permeabilizing the vesicles to varying extents. Electron micrographs of the vesicles after addition of asyn indicate that the enhanced permeability is coupled to large-scale disruption or fusion of the vesicles. These results indicate that asyn is able to permeabilize phospholipid vesicles at low relative concentrations, dependent upon the properties of the vesicles. This could have implications for asyn playing a role in vesicle synthesis, maintenance and fusion within synapses.

Membrane protein structure determination by solid state NMR

In the last 3–5 years, solid state NMR has matured as a method for defining structural details of membrane-embedded proteins and peptides. It is still not, and may never be, a method of choice for complete structural resolution, but the power of the approach for certain systems, and in resolving details for binding sites of ligands and specific parts of proteins, is being proved. As with solution state NMR, stable NMR isotope (2H, 13C, 15N, 19F) incorporation into such large and complex biomolecules has made a tremendous impact in the area, aiding spectral assignments, increasing the amount and value of the information gained, and permitting identification of specific functional intermediates. The practical details of handling membrane proteins, which has for so long dogged every structural and biochemical study of membrane proteins, including crystallography, are not absent in the solid state NMR approach. It is simply that the protein can be studied in situ, or at least in reconstituted systems when the purified or isolated proteins can be reintroduced into a membrane for study. In addition, the molecular weight limit (Mr < 30 000) of solution state NMR (due to the slow molecular tumbling rate with respect to the applied field), is not limiting with solid state methods. Solid state NMR methods can now be used to study membrane proteins and peptides, but support from chemical, molecular biological and biochemical approaches are required to produce systems suitable for giving the desired molecular information. Specialized instrumentation is also required (high power NMR probes with spinning or static capability, specific pulse methods, good temperature stability and wide bore magnets) and some applications can present a challenge experimentally in setting up the NMR instrument. However, the method is emerging as a useful addition for the structural biologist.