Mastoparan binding induces a structural change affecting both the N-terminal and C-terminal domains of calmodulin A 113Cd-NMR study

Abstract113Cd-NMR studies of solutions of cadmium-loaded calmodulin (Cd4CaM) and the tetradecapeptide mastoparan in different ratios show that mastoparan binds to Cd4CaM with high affinity. The off-rate of proteinbound mastoparan is found to be 40 s−1 or less. The binding of one molecule of mastoparan to Cd4CaM is observed to affect all four metal-binding sites, indicating that both the N-terminal and C-terminal globular domains of the protein undergo conformational changes

On the lag phase in amyloid fibril formation.

The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two - primary nucleation and elongation - and in many systems at least four - primary nucleation, elongation, secondary nucleation and fragmentation - microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time.This work was supported by the Swedish Research Council (SL) and its Linneus Centre Organizing Molecular Matter for CD spectrometer, plate readers (SL), the Alzheimer Foundation Sweden (SL), the Frances and Augustus Newman Foundation (TPJK), the BBSRC (TPJK), and the Marie Curie fellowship scheme for career development (PA).This is the final version of the article. It first appeared from RSC via http://dx.doi.org/10.1039/C4CP05563

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D-9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca2+-free protein. In contrast, the linker length has only a marginal effect on the Ca2+ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca2+ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca2+-bound state. For the extreme case of disconnected subdomains, Ca2+ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca2+ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein

Membrane Interaction of α-Synuclein in Different Aggregation States

Aggregated α-synuclein in Lewy bodies is one of the hallmarks of Parkinson's disease (PD). Earlier observations of α-synuclein aggregates in neurons grafted into brains of PD patients suggested cell-to-cell transfer of α-synuclein and a prion-like mechanism. This prompted the current investigation of whether α-synuclein passes over model phospholipid bilayers. We generated giant unilamellar vesicles (GUVs) containing a small amount of a lipid-conjugated red emitting dye (rhodamine B) and varied the membrane charge by using different molar ratios of DOPC and DOPS or cardiolipin. We then used confocal fluorescence microscopy to examine how monomer, fibril as well as on-pathway α-synuclein species labeled with a green emitting fluorophore (Alexa488) interacted with the phospholipid bilayers of the GUV. We defined conditions that yielded reproducible aggregation kinetics under basal conditions and with none or moderate shaking. We found that on-pathway α-synuclein species and equilibrium amyloid aggregates, but not α-synuclein monomers, bound to lipid membranes. α-Synuclein was particularly strongly associated with GUVs containing the anionic lipids cardiolipin or DOPS, whereas it did not associate with GUVs containing only zwitterionic DOPC. We found that α-synuclein progressively aggregated at the surface of the GUVs, typically in distinct domains rather than uniformly covering the membrane, and that both lipid and protein were incorporated in the aggregates. Importantly, we never observed transport of α-synuclein over the GUV bilayer. This suggests that α-synuclein transport over membranes requires additional molecular players and that it might rely on active transport

Extreme Sequence Divergence but Conserved Ligand-Binding Specificity in Streptococcus pyogenes M Protein

Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR) of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP), a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function

Charge Regulation during Amyloid Formation of α-Synuclein

[Image: see text] Electrostatic interactions play crucial roles in protein function. Measuring pK(a) value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pK(a) value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pK(a)(ave) value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pK(a)(ave) values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein

Food Chain Transport of Nanoparticles Affects Behaviour and Fat Metabolism in Fish

Nano-sized (10−9–10−7 m) particles offer many technical and biomedical advances over the bulk material. The use of nanoparticles in cosmetics, detergents, food and other commercial products is rapidly increasing despite little knowledge of their effect on organism metabolism. We show here that commercially manufactured polystyrene nanoparticles, transported through an aquatic food chain from algae, through zooplankton to fish, affect lipid metabolism and behaviour of the top consumer. At least three independent metabolic parameters differed between control and test fish: the weight loss, the triglycerides∶cholesterol ratio in blood serum, and the distribution of cholesterol between muscle and liver. Moreover, we demonstrate that nanoparticles bind to apolipoprotein A-I in fish serum in-vitro, thereby restraining them from properly utilising their fat reserves if absorbed through ingestion. In addition to the metabolic effects, we show that consumption of nanoparticle-containing zooplankton affects the feeding behaviour of the fish. The time it took the fish to consume 95% of the food presented to them was more than doubled for nanoparticle-exposed compared to control fish. Since many nano-sized products will, through the sewage system, end up in freshwater and marine habitats, our study provides a potential bioassay for testing new nano-sized material before manufacturing. In conclusion, our study shows that from knowledge of the molecular composition of the protein corona around nanoparticles it is possible to make a testable molecular hypothesis and bioassay of the potential biological risks of a defined nanoparticle at the organism and ecosystem level

3D MAS NMR Experiment Utilizing Through-Space

We demonstrate a novel 3D NNC magic angle spinning NMR experiment that generates ¹⁵N–¹⁵N internuclear contacts in protein systems using an optimized ¹⁵N–¹⁵N proton assisted recoupling (PAR) mixing period and a ¹³C dimension for improved resolution. The optimized PAR condition permits the acquisition of high signal-to-noise 3D data that enables backbone chemical shift assignments using a strategy that is complementary to current schemes. The spectra can also provide distance constraints. The utility of the experiment is demonstrated on an M₀Aβ₁₋₄₂ fibril sample that yields high-quality data that is readily assigned and interpreted. The 3D NNC experiment therefore provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-001960)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026

A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal beta Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface.

Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the beta protein of group B streptococci. The proline-rich region in beta, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a beta mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the beta protein. Comparison of the XPZ region in different beta proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen

Amyloid-β peptide 37, 38 and 40 individually and cooperatively inhibit amyloid-β 42 aggregation

The pathology of Alzheimer's disease is connected to the aggregation of β-amyloid (Aβ) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, Aβ37 and Aβ38, as pure peptides and in mixtures with Aβ40 and Aβ42. A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform Aβ42. Through kinetic analysis, we show that both Aβ37 and Aβ38 self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar β-sheet-rich aggregates, albeit on a longer timescale than Aβ40 or Aβ42. Additionally, we show that the shorter alloforms co-aggregate with Aβ40, affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither Aβ37 nor Aβ38 forms co-aggregates with Aβ42; however, both short alloforms reduce the rate of Aβ42 aggregation in a concentration-dependent manner. Finally, we show that the aggregation of Aβ42 is more significantly impeded by a combination of Aβ37, Aβ38, and Aβ40 than by any of these alloforms independently. These results demonstrate that the aggregation of any given Aβ alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain. This journal i