Structural insights into differences in G protein activation by family A and family B GPCRs.

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-G protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β adrenergic receptor (βAR; family A).

The Use of Surfactants to Solubilise a Glucagon Analogue.

Purpose: The peptide hormone glucagon, used to treat hypoglycaemic incidents, is prone to aggregation. Generating alternatives with better stability is of pharmaceutical interest in the treatment of diabetes. Here we investigate the impact of six different surfactants on the solubility and stability of ZP-GA-1, a stable version of glucagon.

Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State.

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation.

Large-Scale Biophysical Evaluation of Protein PEGylation Effects: In Vitro Properties of 61 Protein Entities.

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS).

Wildtype and A30P mutant alpha-synuclein form different fibril structures.

Parkinson's Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN.

Multiple roles of heparin in the aggregation of p25α.

The 219-residue protein p25α stimulates the fibrillation of α-synuclein (αSN) in vitro and colocalizes with it in several α-synucleinopathies. Although p25α does not fibrillate by itself under native conditions in vitro, αSN-free p25α aggregates have also been observed in vivo in, for example, multiple system atrophy. To investigate which environmental conditions might trigger this aggregation, we investigated the effect of polyanionic biomolecules on p25α aggregation.

Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation.

One of the major hallmarks of Parkinson disease is aggregation of the protein α-synuclein (αSN). Aggregate cytotoxicity has been linked to an oligomeric species formed at early stages in the aggregation process. Here we follow the fibrillation process of αSN in solution over time using small angle X-ray scattering and resolve four major coexisting species in the fibrillation process, namely monomer, dimer, fibril and an oligomer.

Assays for α-synuclein aggregation.

This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, α-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation.

SDS-induced fibrillation of alpha-synuclein: an alternative fibrillation pathway.

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy.

Strategies to increase the reproducibility of protein fibrillization in plate reader assays.

There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in PD and notorious for its erratic fibrillization behavior. Salts and polyanionic polymers do not significantly improve the quality of the assay.

The influence of vesicle size and composition on alpha-synuclein structure and stability.

Monomeric alpha-synuclein (alphaSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in alpha-helix structure. Here we show that at physiological pH and ionic strength, alphaSN incubated with different anionic lipid vesicles undergoes a marked increase in alpha-helical content at a temperature dictated either by the temperature of the lipid phase transition, or (in 1,2-DilauroylSN-Glycero-3-[Phospho-rac-(1-glycerol)] (DLPG), which is fluid down to 0 degrees C) by an intrinsic cold denaturation that occurs around 10-20 degrees C. This structure is subsequently lost in a thermal transition around 60 degrees C.

An unusual intrinsically disordered protein from the model legume Lotus japonicus stabilizes proteins in vitro.

Intrinsic structural disorder is a prevalent feature of proteins with chaperone activity. Using a complementary set of techniques, we have structurally characterized LjIDP1 (intrinsically disordered protein 1) from the model legume Lotus japonicus, and our results provide the first structural characterization of a member of the Lea5 protein family (PF03242). Contrary to in silico predictions, we show that LjIDP1 is intrinsically disordered and probably exists as an ensemble of conformations with limited residual beta-sheet, turn/loop, and polyproline II secondary structure.

Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions.

Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect.

Aggregation as the basis for complex behaviour of cutinase in different denaturants.

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate.