Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils.

Many proteins and peptides self-associate into highly ordered and structurally similar amyloid cross-ß aggregates. This fibrillation is critically dependent on properties of the protein and the surrounding environment that alter kinetic and thermodynamic equilibria. Here, we report on dominating surface and solution effects on the fibrillogenic behavior and amyloid assembly of the C-36 peptide, a circulating bioactive peptide from the α1-antitrypsin serine protease inhibitor. C-36 converts from an unstructured peptide to mature amyloid twisted-ribbon fibrils over a few hours when incubated on polystyrene plates under physiological conditions through a pathway dominated by surface-enhanced nucleation. In contrast, in plates with nonbinding surfaces, slow bulk nucleation takes precedence over surface catalysis and leads to fibrillar polymorphism. Fibrillation is strongly ion-sensitive, underlining the interplay between hydrophilic and hydrophobic forces in molecular self-assembly. The addition of exogenous surfaces in the form of silica glass beads and polyanionic heparin molecules potently seeds the amyloid conversion process. In particular, heparin acts as an interacting template that rapidly forces ß-sheet aggregation of C-36 to distinct amyloid species within minutes and leads to a more homogeneous fibril population according to solid-state NMR analysis. Heparin's template effect highlights its role in amyloid seeding and homogeneous self-assembly, which applies both in vitro and in vivo, where glycosaminoglycans are strongly associated with amyloid deposits. Our study illustrates the versatile thermodynamic landscape of amyloid formation and highlights how different experimental conditions direct C-36 into distinct macromolecular structures.

Coexistence of ribbon and helical fibrils originating from hIAPP(20-29) revealed by quantitative nanomechanical atomic force microscopy.

Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP(20-29), which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP(20-29) nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP(20-29) shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing ß sheets in cross-ß motifs as the basis of self-assembled amyloids.

Diffusion-weighted MRI and quantitative biophysical modeling of hippocampal neurite loss in chronic stress.

Chronic stress has detrimental effects on physiology, learning and memory and is involved in the development of anxiety and depressive disorders. Besides changes in synaptic formation and neurogenesis, chronic stress also induces dendritic remodeling in the hippocampus, amygdala and the prefrontal cortex. Investigations of dendritic remodeling during development and treatment of stress are currently limited by the invasive nature of histological and stereological methods. Here we show that high field diffusion-weighted MRI combined with quantitative biophysical modeling of the hippocampal dendritic loss in 21 day restraint stressed rats highly correlates with former histological findings. Our study strongly indicates that diffusion-weighted MRI is sensitive to regional dendritic loss and thus a promising candidate for non-invasive studies of dendritic plasticity in chronic stress and stress-related disorders.

Fast mapping of global protein folding states by multivariate NMR: a GPS for proteins.

To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method that provides such an overview. GPS NMR exploits the unique ability of NMR to simultaneously record signals from individual hydrogen atoms in complex macromolecular systems and of multivariate analysis to describe spectral variations from these by a few variables for establishment of, and positioning in, protein-folding state maps. The method is fast, sensitive, and robust, and it works without isotope-labelling. The unique capabilities of GPS NMR to identify different folding states and to compare different unfolding processes are demonstrated by mapping of the equilibrium folding space of bovine alpha-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat.

Metabolic changes during estivation in the common earthworm Aporrectodea caliginosa.

The common earthworm Aporrectodea caliginosa survives drought by forming estivation chambers in the topsoil under even very slight reductions in soil water activity. We induced estivation in a soil of a consistency that allowed the removal of intact soil estivation chambers containing a single worm. These estivation chambers were exposed to 97% relative humidity for 30 d to simulate the effect of a severe summer drought. Gas exchange, body fluid osmolality, water balance, urea, and alanine were quantified, and whole-body homogenates were screened for changes in small organic molecules via (1)H-nuclear magnetic resonance (NMR). Formation of estivation chambers was associated with a dramatic increase in body fluid osmolality, from 175 to 562 mOsm kg(-1), accompanied by a 20% increase in water content. Dehydration for 1 mo caused a further increase to 684 mOsm kg(-1), while the worms lost 50% of their water content. Gas exchange was depressed by 50% after worms entered estivation and by 80% after a further 30 d of dehydration. Urea concentrations increased from 0.3 to 1 micromol g(-1) dry mass during this time. Although (1)H-NMR did not provide the identity of the osmolytes responsible for the initial increase in osmolality after estivation, it showed that alanine increased to more than 80 mmol L(-1) in the long-term-estivation group. We propose that alanine functions as a nitrogen depot during dehydration and is not an anaerobe product in this case.

Unique identification of supramolecular structures in amyloid fibrils by solid-state NMR spectroscopy.

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 A resolution. Symmetry information contained in the easily obtainable resonance assignments from solid-state NMR spectra (see picture), along with long-range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

The folding of human active and inactive extracellular superoxide dismutases is an intracellular event.

Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.

An expedient synthesis of the fibril binding compound FSB via sequential Pd-catalyzed coupling reactions.

The styryl benzene derivative (E, E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), well-known for its binding to beta-amyloid peptide fibrils, was synthesized in an efficient manner exploiting two sequential palladium(0)-catalyzed coupling reactions in a 34% overall yield. This is a substantial improvement to the previously reported synthesis of FSB in 1.1%.

Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed.

A mutant Bowman-Birk gene was created that encoded an inactive high-sulfur product. It was used to transform soybean line Asgrow 3237. Transformants bearing the mutant gene were identified by GUS expression, PCR analysis, and Southern analysis. The amount of steady state mRNA from the mutant gene in the transformed plants showed that the gene was highly expressed, but the amount of message from the unmodified Bowman-Birk gene did not change detectably. Proteins synthesized at the direction of the mutant Bowman-Birk gene accumulated in seeds of the transformed plants, and there was a marked decrease in the ability of extracts prepared from these seeds to inhibit trypsin and chymotrypsin despite the presence of Kunitz trypsin inhibitor. The more prevalent mRNA from the mutant gene was considered to out-compete message from the native genes to decrease the amount of active Bowman-Birk inhibitor.

Socioeconomic correlates of drug use based on prescription data: a population-based cross-sectional register study in Denmark 1999.

INTRODUCTION: In the public health system we study medical treatment which is ideally provided according to need and independently of economic means. We report use of prescription drugs according to socioeconomic classifications in North Jutland County in Denmark in 1999. METHOD: We conducted a register-based cross-sectional study of 385,879 persons aged 18 years or older. Data from the computerized accounting system from the pharmacies were linked with records of socioeconomic status (SES) in the Prevention Registry at Statistics Denmark. We identified all prescriptions redeemed in North Jutland County from 1 January through 31 December 1999 and classified the socioeconomic status for each individual based on the annual registration of income/social benefits, employer, occupation and education. We computed the proportion of persons redeeming at least one prescription and computed weighted averages of prescription proportions for each SES. RESULTS: The highest prevalence of medication use was by persons in early retirement, old age pensioners, people on disability pension and others outside the workforce. We found only minor differences among different economically active groups with slightly more male top managers using cardiovascular drugs. People in the upper half of the socioeconomic scale were somewhat less likely to redeem prescriptions for treating muscle, joints and bone, and central nervous system. CONCLUSION: Social or economic barriers in buying medicine are generally small in Denmark and do probably not provide a likely explanation for the social differences in morbidity and mortality.

Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli.

DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.

Improved pulse sequences for pure exchange solid-state NMR spectroscopy.

Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin.