Gerhard Gröbner

How is protein aggregation in amyloidogenic diseases modulated by biological membranes

The fate of proteins with amyloidogenic properties depends critically on their immediate biochemical environment. However, the role of biological interfaces such as membrane surfaces, as promoters of pathological aggregation of amyloidogenic proteins, is rarely studied and only established for the amyloid-β protein (Aβ) involved in Alzheimer’s disease, and α-synuclein in Parkinsonism. The occurrence of binding and misfolding of these proteins on membrane surfaces, is poorly understood, not at least due to the two-dimensional character of this event. Clearly, the nature of the folding pathway for Aβ protein adsorbed upon two-dimensional aggregation templates, must be fundamentally different from the three-dimensional situation in solution. Here, we summarize the current research and focus on the function of membrane interfaces as aggregation templates for amyloidogenic proteins (and even prionic ones). One major aspect will be the relationship between membrane properties and protein association and the consequences for amyloidogenic products. The other focus will be on a general understanding of protein folding pathways on two-dimensional templates on a molecular level. Finally, we will demonstrate the potential importance of membrane-mediated aggregation for non-amphiphatic soluble amyloidogenic proteins, by using the SOD1 protein involved in the amyotrophic lateral sclerosis syndrome.

Observations of light-induced structural changes of retinal within rhodopsin

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 Å) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated MI state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

Biochemical and functional characterization of Helicobacter pylori vesicles.

Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl‐Lewis x/a (sLex/a) antigens. Similar to most Gram‐negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence‐associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle–host interactions and found that the vesicles carry effector‐promoting properties that are important to disease development.

Detergent-free formation and physicochemical characterization of nanosized lipid-polymer complexes: Lipodisq.

Lipodisq particles are polymer-lipid complexes formed by detergent-free methods. Lipodisq particles containing dimyristoylphosphatidylcholine (DMPC) are characterized by increased lipid ordering co ...

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

The dehydrin family of proteins is upregulated in response to desiccation and cold stress and is thought to protect the plant by stabilizing the plasma membrane. This study characterizes the interaction between a typical dehydrin (Lti30) and lipid membranes. Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30’s proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest.

Chitosan in situ gelation for improved drug loading and retention in poloxamer 407 gels

A method for the in situ gelation of poloxamers and the mucoadhesive polymer chitosan has been developed by exploiting the tendency of poloxamer solution to form gel at physiological temperatures and of chitosan (CT) to form ionotropic gel structures in the presence of sodium tripolyphosphate (TPP). Novel poloxamer gels containing CT-TPP complex formed in situ during the administration were prepared by mixing poloxamer-CT and poloxamer-TPP solutions in double syringes. The micellization and gelation of poloxamer 407 in the presence of chitosan and/or TPP were studied using differential scanning calorimetry and tube inversion; both additives were found to reduce the critical micellization temperature and critical gelation temperature of poloxamer aqueous solution. The poloxamer gels containing CT-TPP complex formed in situ were found to exhibit reduced dissolution rate and superior release characteristics with three different drugs--metoprolol, doxycycline and flufenamic acid. Furthermore, by varying the compositions of the two solutions independently, it is possible to control the pH in a way to suit the solubilization of a drug as well as the specific environment of a particular application site. By varying the concentrations of chitosan, TPP and poloxamer, the delivery system can be fine-tuned to afford gels with specific properties, ranging from nanoparticle suspensions to semisolid gels. These in situ gels have the potential to increase the utility of thermo-reversible poloxamers in drug delivery.

High-resolution characterization of organic phosphorus in soil extracts using 2D 1H-31P NMR correlation spectroscopy.

Organic phosphorus (P) compounds represent a major component of soil P in many soils and are key sources of P for microbes and plants. Solution NMR (nuclear magnetic resonance spectroscopy) is a powerful technique for characterizing organic P species. However, (31)P NMR spectra are often complicated by overlapping peaks, which hampers identification and quantification of the numerous P species present in soils. Overlap is often exacerbated by the presence of paramagnetic metal ions, even if they are in complexes with EDTA following NaOH/EDTA extraction. By removing paramagnetic impurities using a new precipitation protocol, we achieved a dramatic improvement in spectral resolution. Furthermore, the obtained reduction in line widths enabled the use of multidimensional NMR methods to resolve overlapping (31)P signals. Using the new protocol on samples from two boreal humus soils with different Fe contents, 2D (1)H-(31)P correlation spectra allowed unambiguous identification of a large number of P species based on their (31)P and (1)H chemical shifts and their characteristic coupling patterns, which would not have been possible using previous protocols. This approach can be used to identify organic P species in samples from both terrestrial and aquatic environments increasing our understanding of organic P biogeochemistry.

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Aβ1−40 peptide in solid‐supported lipid bilayers

We utilized plasmon‐waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid β peptide1−40 in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of ≈ 2.5‐fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high‐resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM‐rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC‐enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mm zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.

SOD1 Mutations Targeting Surface Hydrogen Bonds Promote Amyotrophic Lateral Sclerosis without Reducing Apo-state Stability

In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

Disordered Proteins: Biological Membranes as Two-Dimensional Aggregation Matrices

Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-β protein (Aβ), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane–protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.