A family of neurodegenerative diseases, including Huntington’s disease (HD) and spinocerebellar ataxias, are associated with an abnormal polyglutamine (polyQ) expansion in mutant proteins that become ...prone to form amyloid-like aggregates. Prior studies have suggested a key role for β-hairpin formation as a driver of nucleation and aggregation, but direct experimental studies have been challenging. Toward such research, we set out to enable spatiotemporal control over β-hairpin formation by the introduction of a photosensitive β-turn mimic in the polypeptide backbone, consisting of a newly designed azobenzene derivative. The reported derivative overcomes the limitations of prior approaches associated with poor photochemical properties and imperfect structural compatibility with the desired β-turn structure. A new azobenzene-based β-turn mimic was designed, synthesized, and found to display improved photochemical properties, both prior and after incorporation into the backbone of a polyQ polypeptide. The two isomers of the azobenzene-polyQ peptide showed different aggregate structures of the polyQ peptide fibrils, as demonstrated by electron microscopy and solid-state NMR (ssNMR). Notably, only peptides in which the β-turn structure was stabilized (azobenzene in the cis configuration) closely reproduced the spectral fingerprints of toxic, β-hairpin-containing fibrils formed by mutant huntingtin protein fragments implicated in HD. These approaches and findings will enable better deciphering of the roles of β-hairpin structures in protein aggregation processes in HD and other amyloid-related neurodegenerative diseases.
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•A mitochondrial protein-lipid complex regulates lipid peroxidation in apoptosis.•Peroxidase-active lipid-cytochrome c complexes are reconstituted in vitro.•Phosphatidylglycerol ...lipids are less effective activators than cardiolipin.•Activity correlates to protein dynamics that are distinct from full denaturation.•A dynamic interplay of cytochrome c foldons and anionic lipids regulate activity.
Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.
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The stability of lactate dehydrogenase (LDH) and β-galactosidase (β-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. ...The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while β-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, β-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and β-gal may be dependent on the interaction with arginine.
Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density ...functional theory (DFT). We measured the chemical shifts of the N-formyl-l-methionyl-l-leucyl-l-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the 13C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that 13C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 μs classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.
Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ ...complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.
We employ a combination of 13C/15N magic angle spinning (MAS) NMR and 2H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the ...effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. 2H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. 2H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ∼27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.
The lipid bilayer typical of hydrated biological membranes is characterized by a liquid-crystalline, highly dynamic state. Upon cooling or dehydration, these membranes undergo a cooperative ...transition to a rigidified, more-ordered, gel phase. This characteristic phase transition is of significant biological and biophysical interest, for instance in studies of freezing-tolerant organisms. Magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy allows for the detection and characterization of the phase transitions over a wide temperature range. In this study we employ MAS 1H NMR to probe the phase transitions of both solvent molecules and different hydrated phospholipids, including tetraoleoyl cardiolipin (TOCL) and several phosphatidylcholine lipid species. The employed MAS NMR sample conditions cause a previously noted substantial reduction in the freezing point of the solvent phase. The effect on the solvent is caused by confinement of the aqueous solvent in the small and densely packed MAS NMR samples. In this study we report and examine how the freezing point depression also impacts the lipid phase transition, causing a ssNMR-observed reduction in the lipids’ melting temperature (Tm). The molecular underpinnings of this phenomenon are discussed and compared with previous studies of membrane-associated water phases and the impact of membrane-protective cryoprotectants.
At reduced temperatures, proteins and other biomolecules are generally found to exhibit dynamic as well as structural transitions. This includes a so-called protein glass transition that is ...universally observed in systems cooled between 200 and 230 K, and which is generally attributed to interactions between hydrating solvent molecules and protein side chains. However, there is also experimental and theoretical evidence for a low-temperature transition in the intrinsic dynamics of the protein itself, absent any solvent. Here, we use low-temperature solid-state NMR to examine site-specific fluctuations in atomic structure and dynamics in the absence of solvents. In particular, we employ magic angle spinning NMR to examine a structural phase transition associated with dynamic processes in a solvent-free polypeptide, N-f-MLF-OH, lattice at temperatures as low as 90 K. This transition is characterized by the appearance of an extra set of lines in 1D 15N spectra as well as additional cross peaks in 2D 13C−13C and 13C−15N spectra. Interestingly, the gradual, temperature-dependent appearance of the new spectral component is not accompanied by the line broadening typical of dynamic transitions. A direct comparison between the spectra of N-f-MLF-OH and the analog N-f-MLF-OMe, which does not display this transition, indicates a correlation of the structural transition to the temperature dependent motion of the aromatic phenylalanine side chain. Several quantitative solid state NMR experiments were employed to provide site-specific measurements of structural and motional features of the observed transition.
Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's ...disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.
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•d-PolyQ amyloid fibrils are equipotent with l-fibrils in killing cells in culture.•d-PolyQ fibrils seed aggregation of l-polyQ monomers in cells and in vitro.•Achiral cross-seeding can be rationalized by a d,l rippled β-sheet interface.•The results have implications for molecular mechanisms of Huntington's disease.•Relaxed stringency in amyloid propagation also has implications for prion biology.
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