Michler’s hydrol blue elucidates structural differences in prion strains

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Amyloids are unbranched fibrils of protein aggregates arranged as β-strands that run perpendicular to the fiber axis, forming a cross β-sheet of indefinite length (1–4). They are related to several common human diseases such as Alzheimer’s disease and type II diabetes (4–7). Amyloids can also be beneficial, such as those found in yeast, where they can help cells survive environmental fluctuations (6, 8, 9). An important feature of amyloid fibrils is their polymorphism, which refers to structural variations in fibrils formed by a particular polypeptide chain under the same environmental conditions (2–4, 6, 10–16). In yeast, as well as in other organisms, polymorphisms are also referred to as strains because cells that harbor them have different phenotypes (17, 18). The yeast prion protein, Sup35, can adopt a variety of amyloid polymorphs, of which at least two, named “weak” and “strong,” describe the phenotypes they confer in vivo (19). The amyloid core of the strong prion fibrils comprises the first 40 residues of Sup35, and the amyloid core of the weak prion fibrils comprises the first 70 residues (17, 20). These two polymorphs have tightly packed β-sheet-rich amyloid cores with distinct molecular structures (20, 21) that are protected from the outside environment. However, the location of the beta strands and turns for individual monomers of Sup35 when they are assembled into any amyloid form remains poorly defined since Sup35 amyloid fibers have highly degenerate sequences, making them difficult to study by solid-state NMR spectroscopy, and lack repeating structural features, making them difficult to study using cryo-electron microscopy.The most commonly used probe to monitor amyloid fibril formation is still thioflavin T (ThT), a fluorescent molecular rotor discovered many decades ago (22–25). When bound to amyloid fibrils, molecular rotors become conformationally confined and experience an environment with different polarity compared to bulk water. The conformational restriction increases the quantum efficiency of the molecular rotors, making these dyes sensitive amyloid indicators and thus particularly suitable for amyloid fibril detection. However, the spectral properties of ThT, as well as other classic amyloid dyes such as Congo red, are not altered by changes in polarity, and thus, they do not distinguish between different amyloid polymorphs (26). Some more modern amyloid dyes, such as luminescent-conjugated polythiophenes, can distinguish between amyloid polymorphs; however, the resulting spectral changes cannot be directly attributed to any specific differences in the physiochemical properties of the different fibril structures (27–29). Michler’s hydrol blue (MHB), a symmetric molecular rotor, binds to amyloid fibrils with high affinity, and the spectral properties of MHB are sensitive to local changes in hydrophobicity (30). MHB aligns parallel to the fiber at an angle of 14° to 22° to the axis along the grooves of the β-sheets and preferentially binds near aromatic rings (30). Quantum mechanical investigations explain this valuable property of MHB, and the related group of cationic tri- and diarylmethane dyes (31–34); the lowest excited state dynamics in the two lowest excited states of MHB are sensitive to the polarity of the local environment. As some of the features for MHB binding to amyloid fibers are defined and the spectral properties of the dye are dependent on the local environment, MHB could potentially be a structurally specific fluorescent probe.In this work, we use fluorescence spectroscopy to monitor amyloid assembly using the molecular rotor dye MHB. We use computational simulations to determine whether and how the change in hydrophobicity associated with a tyrosine to phenylalanine mutation could alter the fluorescence properties of MHB. We test these predictions on a series of weak and strong prion fibrils harboring single tyrosine to phenylalanine mutations and report the excitation and emission spectra of MHB in the presence of Sup35NM in the weak and strong prion fibril conformations. We find that MHB displays different spectral shifts when bound to the strong and weak prions. Furthermore, the analysis of Sup35 mutants in which a tyrosine in the N-terminal region was replaced by phenylalanine shows that the probe can detect differences in the structure and polarity of the fibrils that can be localized to a specific residue.     

A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) contributes to the expression of familial combined hyperlipidemia

We performed denaturing gradient gel electrophor-esis (DGGE)of exons 4, 5, 6 and their exon-intron boundaries of the LPL-genein 169 unrelated male patients suffering from familial combinedhyperlipide-mia (FCH). Twenty patients were found to carry anucleotide substitution in exon 6. Sequence and PCR/ digestionanalysis revealed one common mutation (Asn291Ser) in all thesecases. This mutation was also present in 215 male controls,albeit at a lower frequency than in FCH patients (10/215 = 4.6% vs. 20/ 169 = 11. 8% p <0. 02). Analysis of lipid, lipoproteinand apolipoprotein levels demonstrated an association betweenthe presence of this Asn291Ser substitution and decreased HDL-cholesterol(0. 94 ± 0. 31 vs. 1. 12 ± 0. 26 mmol/ l; p <0. 04) in our controls. FCH patients carrying this mutationshowed decreased HDL-cholesterol (0.75 ± 0. 16 vs. 0.95 ± 0.36 mmol/l; p = 0. 05) and increased triglyceridelevels (5. 96 ± 4. 12 vs. 3.48 ± 1.78 mmol/ l;p < 0. 005) compared to non-carriers. The high triglycerideand low HDL-cholesterol phenotype in carriers of this substitutionwas most obvious when BMI exceded 27 kg/ m². Our study of maleFCH patients revealed the presence of a common mutation in theLPL-gene that is associated with lipoprotein abnormalities,indicating that defective LPL is at least one of the factorscontributing to the FCH-phenotype.     

Orientation of aromatic residues in amyloid cores: Structural insights into prion fiber diversity

Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing.Amyloids comprise a diverse group of protein polymers characterized by beta-strands that run perpendicular to the polymeric fiber axis. They are associated with devastating economic hardship in an extraordinary variety of settings—ranging from the degenerative diseases of our aging population (1) to the bacterial biofilms that resist eradication by antibiotics, bacteriophage, and even bleach (2). However, amyloids also provide beneficial functions; for example, they help to maintain long-term neuronal synapses (3, 4). Amyloid proteins are the structural basis for a paradigm shift in microbial genetics: Conformational changes of self-templating amyloids form protein-based elements of inheritance, known as “prions” (5–7), that create phenotypic diversity in changing environments (8, 9). A peculiar, and still mysterious, property of prions (and virtually all amyloidogenic proteins) is the ability of the same polypeptide chain to stably adopt distinct amyloid folds with different physical and biological properties (10–12). These are referred to as prion “strains” and are named for the distinct biological phenotypes they confer. Amyloid strains were first described for the mammalian prion protein PrP, which is responsible for transmissible spongiform encephalopathy (13, 14). They now seem to be a general property of amyloids associated with various neurodegenerative diseases (15–18). Indeed, many of these have prion-like self-templating dispersion properties in vivo that are associated with different disease phenotypes (19–21).However, despite the importance of amyloids in so many aspects of biology, the amyloid fold remains one of the most poorly understood of all basic protein folds. This is mainly because the methods for structural characterization of such insoluble polymers are limited. Here, by using a structural probe of amyloid fibers and two mechanisms of fiber orientation, we demonstrate the utility of polarized-light spectroscopy measurements (linear dichroism, LD) to determine accurate angular data of aromatic side groups in amyloid fibers. We apply this method to amyloid fibers of the yeast prion protein Sup35, the translation termination factor in yeast. Sup35 has three functional domains: an amyloid forming amino-terminal domain (N), a highly charged middle domain (M), and a carboxyl-terminal domain (C), which is involved in translation termination. Tyrosines are well distributed in the amyloid-forming domain of the protein, providing plentiful structural probes in the amyloid core (Fig. 1A). The N and M domains (Sup35NM) are responsible for the prion activity. When Sup35 switches from its native conformation to an amyloid form, the fidelity of translation termination changes and new phenotypes are created (9). Upon conversion into a prion state Sup35NM can adopt a variety of distinct amyloid-rich fiber conformations. There are at least two fiber forms of Sup35NM, called “weak” and “strong” for the phenotypes they confer in vivo rather than for their biophysical properties (10). For example, amyloids that confer a strong phenotype are biophysically more fragile. More Sup35 is therefore sequestered in the amyloid form owing to an increase in fiber ends, resulting in stronger stop-codon read-through phenotypes in vivo. The amino acids that control the conformational switch have been delineated but their structural constraints are only loosely defined (22–25). LD, defined as the differential absorption between light polarized, parallel and perpendicular to a macroscopic orientation direction, revealed a general structural feature of amyloids. Moreover, the method is sensitive enough to characterize structural variations between strains.Open in a separate windowFig. 1.Principles of polarized light measurements. (A) Sequence of NM domain (residues 1–253) of yeast Sup35 prion protein. N domain is shown in blue with tyrosine (Y) residues (whose orientation is detected by LD) highlighted with yellow. (B) Transition dipole moments of UV transitions L_a and L_b of tyrosine chromophore. (C and D) The two alignment techniques used in the study: orientation in a stretched film (C) and in a Couette shear flow cell (D).     

From the Cover: Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.     

The Asn9 variant of lipoprotein lipase is associated with the — 93G promoter mutation and an increased risk of coronary artery disease

Two mutations in the lipoprotein lipase (LPL) gene, a T to G transition at position −93 of the proximal promoter region and an Asp9Asn substitution in exon 2, were examined in 762 Dutch males with angiographically-diagnosed coronary artery disease (CAD) and 296 healthy normolipidemic Dutch males. The two mutations exhibited strong linkage disequilibrium (D'=0.975). A significantly higher proportion of cases (4.86%) than controls (1.37%) carried the −93G/Asn9 allele (p=0.008). In the combined sample of cases and controls, adjusted mean plasma total cholesterol (TC) levels were significantly higher in −93G/Asn9 carriers (6.20±0.13 mmol/l) than in non-carriers (5.93±0.03 mmol/l; p=0.048), while mean high-density lipoprotein cholesterol (HDL-C) levels were lower in carriers (0.88±0.03 mmol/l) than in non-carriers (0.98±0.01 mmol/l; p=0.002). There was a trend towards higher triglyceride (TG) levels in carriers (1.96±0.14 mmol/l) compared with non-carriers (1.73±0.03 mmol/l) (p=0.08). Additionally, carrier frequencies in tertiles of TC, HDL-C, TG, and LPL activity, suggested an association of the −93G/Asn9 variant with higher TC and TG levels, and with lower HDL-C and LPL activity levels. Logistic regression revealed a significant odds ratio (OR) for the combined −93G/Asn9 genotype in CAD cases relative to controls (OR: 5.36; 95% CI: 1.57–18.24), with age, body mass index (BMI), smoking, and plasma total- and HDL-cholesterol levels included in the model. In conclusion, we show that the LPL Asp9Asn mutation is in non-random association with a T→G substitution at position −93 of the proximal promoter region and that the combined −93G/Asn9 genotype predisposes to decreased HDL-C levels and an increased risk of CAD.