Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Crowding Alone Cannot Account for Cosolute Effect on Amyloid Aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed “macromolecular crowding”. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation

Assignment of PolyProline II Conformation and Analysis of Sequence – Structure Relationship

International audienceBACKGROUND: Secondary structures are elements of great importance in structural biology, biochemistry and bioinformatics. They are broadly composed of two repetitive structures namely α-helices and β-sheets, apart from turns, and the rest is associated to coil. These repetitive secondary structures have specific and conserved biophysical and geometric properties. PolyProline II (PPII) helix is yet another interesting repetitive structure which is less frequent and not usually associated with stabilizing interactions. Recent studies have shown that PPII frequency is higher than expected, and they could have an important role in protein - protein interactions. METHODOLOGY/PRINCIPAL FINDINGS: A major factor that limits the study of PPII is that its assignment cannot be carried out with the most commonly used secondary structure assignment methods (SSAMs). The purpose of this work is to propose a PPII assignment methodology that can be defined in the frame of DSSP secondary structure assignment. Considering the ambiguity in PPII assignments by different methods, a consensus assignment strategy was utilized. To define the most consensual rule of PPII assignment, three SSAMs that can assign PPII, were compared and analyzed. The assignment rule was defined to have a maximum coverage of all assignments made by these SSAMs. Not many constraints were added to the assignment and only PPII helices of at least 2 residues length are defined. CONCLUSIONS/SIGNIFICANCE: The simple rules designed in this study for characterizing PPII conformation, lead to the assignment of 5% of all amino as PPII. Sequence - structure relationships associated with PPII, defined by the different SSAMs, underline few striking differences. A specific study of amino acid preferences in their N and C-cap regions was carried out as their solvent accessibility and contact patterns. Thus the assignment of PPII can be coupled with DSSP and thus opens a simple way for further analysis in this field

Enhancing Surface Enhanced Raman Scattering (SERS) detection of propranolol with multiobjective evolutionary optimization

Colloidal-based surface-enhanced Raman scattering (SERS) is a complex technique, where interaction between multiple parameters, such as colloid type, its concentration, and aggregating agent, is poorly understood. As a result SERS has so far achieved limited reproducibility. Therefore the aim of this study was to improve enhancement and reproducibility in SERS, and to achieve this, we have developed a multiobjective evolutionary algorithm (MOEA) based on Pareto optimality. In this MOEA approach, we tested a combination of five different colloids with six different aggregating agents, and a wide range of concentrations for both were explored; in addition we included in the optimization process three laser excitation wavelengths. For this optimization of experimental conditions for SERS, we chose the β-adrenergic blocker drug propranolol as the target analyte. The objective functions chosen suitable for this multiobjective problem were the ratio between the full width at half-maximum and the half-maximum intensity for enhancement and correlation coefficient for reproducibility. To analyze a full search of all the experimental conditions, 7785 experiments would have to be performed empirically; however, we demonstrated the search for acceptable experimental conditions of SERS can be achieved using only 4% of these possible experiments. The MOEA identified several experimental conditions for each objective which allowed a limit of detection of 2.36 ng/mL (7.97 nM) propranolol, and this is significantly lower (>25 times) than previous SERS studies aimed at detecting this β-blocker

Solution structure and dynamics of biomolecules from Raman optical activity.

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies