Protein Folding through Kinetic Discrimination

Proteins fold on a μs−ms time scale. However, the number of possible conformations of the polypeptide backbone is so large that random sampling would not allow the protein to fold within the lifetime of the universe, the Levinthal paradox. We show here that a protein chain can fold efficiently with high fidelity if on average native contacts survive longer than non-native ones, that is, if the dissociation rate constant for breakage of a contact is lower for native than for non-native interactions. An important consequence of this finding is that no pathway needs to be specified for a protein to fold. Instead, kinetic discrimination among formed contacts is a sufficient criterion for folding to proceed to the native state. Successful protein folding requires that productive contacts survive long enough to obtain a certain level of probability that other native contacts form before the first interacting unit dissociates. If native contacts survive longer than non-native ones, this prevents misfolding and provides the folding process with directionality toward the native state. If on average all contacts survive equally long, the protein chain is deemed to fold through random search through all possible conformations (i.e., the Levinthal paradox). A modest degree of cooperativity among the native contacts, that is, decreased dissociation rate next to neighboring contacts, shifts the required ratio of dissociation rates into a realistic regime and makes folding a stochastic process with a nucleation step. No kinetic discrimination needs to be invoked in regards to the association process, which is modeled as dependent on the diffusion rate of chain segments

Ganglioside Micelles Affect Amyloid β Aggregation by Coassembly

Amyloid β peptide (Aβ) is the crucial protein component of extracellular plaques in Alzheimer’s disease. The plaques also contain gangliosides lipids, which are abundant in membranes of neuronal cells and in cell-derived vesicles and exosomes. When present at concentrations above its critical micelle concentration (cmc), gangliosides can occur as mixed micelles. Here, we study the coassembly of the ganglioside GM1 and the Aβ peptides Aβ40 and 42 by means of microfluidic diffusional sizing, confocal microscopy, and cryogenic transmission electron microscopy. We also study the effects of lipid–peptide interactions on the amyloid aggregation process by fluorescence spectroscopy. Our results reveal coassembly of GM1 lipids with both Aβ monomers and Aβ fibrils. The results of the nonseeded kinetics experiments show that Aβ40 aggregation is delayed with increasing GM1 concentration, while that of Aβ42 is accelerated. In seeded aggregation reactions, the addition of GM1 leads to a retardation of the aggregation process of both peptides. Thus, while the effect on nucleation differs between the two peptides, GM1 may inhibit the elongation of both types of fibrils. These results shed light on glycolipid–peptide interactions that may play an important role in Alzheimer’s pathology

Calmodulin Transduces Ca²⁺ Oscillations into Differential Regulation of Its Target Proteins

Diverse physiological processes are regulated differentially by Ca²⁺ oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca²⁺ binding affinities, and target exchange rates that depend on both target identity and Ca²⁺ occupancy. The binding dynamics among Ca²⁺, Mg²⁺, calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca²⁺-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca²⁺ transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin

Fibril Charge Affects α‑Synuclein Hydrogel Rheological Properties

In this paper, we have investigated the interactions between α-synuclein fibrils at different pH values and how this relates to hydrogel formation and gel properties. Using a combination of rheology, small-angle X-ray scattering, Raman spectroscopy, and cryo-transmission electron microscopy (cryo-TEM) experiments, we have been able to investigate the relationship between protein net charge, fibril–fibril interactions, and hydrogel properties, and have explored the potential for α-synuclein to form hydrogels at various conditions. We have found that α-synuclein can form hydrogels at lower concentrations (50–300 μM) and over a wider pH range (6.0–7.5) than previously reported. Over this pH range and at 300 μM, the fibril network is electrostatically stabilized. Decreasing the pH to 5.5 results in the precipitation of fibrils. A maximum in gel stiffness was observed at pH 6.5 (∼1300 Pa), which indicates that significant attractive interactions operate at this pH and cause an increase in the density of hydrophobic contacts between the otherwise negatively charged fibrils. We conclude that fibril–fibril interactions under these conditions involve both long-range electrostatic repulsion and a short-range hydrophobic attractive (sticky) component. These results may provide a basis for potential applications and add to the understanding of amyloids

Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (<i>t</i>_1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration

Effects of Polyamino Acids and Polyelectrolytes on Amyloid β Fibril Formation

The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly­(acrylic acid), poly­(ethylenimine), and poly­(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide

Residue-Specific p<i>K</i>_a Determination of Lysine and Arginine Side Chains by Indirect ¹⁵N and ¹³C NMR Spectroscopy: Application to <i>apo</i> Calmodulin

Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants. We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pKa determination of individual amino acid side chains. The experiments follow the side-chain 15Nζ (lysine) and 15Nε or 13Cζ (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede 1H-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the 15Nζ and 15Nε nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described:  apo calmodulin, calbindin D9k, and FKBP12. For apo calmodulin, residue-specific pKa values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification

The Binding Site for Human C4BP in the Hypervariable Region (HVR) of M Protein

<div><p>(A) Schematic representation of C4BP bound to the HVR of an M protein, a dimeric coiled-coil. The most common form of C4BP has seven identical α-chains and one short β-chain. Both chains are composed of CCP modules, as indicated. The binding site for M protein in C4BP is located in the CCP1–2 region of the α-chain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b017" target="_blank">17</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b047" target="_blank">47</a>].</p><p>(B) Multiple sequence alignment of HVRs that bind C4BP. The five upper sequences are from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b025" target="_blank">25</a>]. Three residues that are identical in these five sequences are boxed. PrtH is a second M protein expressed by certain M1 strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b035" target="_blank">35</a>]. The lower part of the alignment shows the HVRs of M4.1 and M114, characterized in this paper. The vertical hatched lines, corresponding to residues 1–39 in M22, indicate the region used to generate the logo in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g005" target="_blank">Figure 5</a>A.</p><p>(C) Construction of fusion proteins derived from the M22 and M5 proteins. An N-terminal region derived from M22 was fused to the C-terminal part of M5 (residues 104–450 of M5). The fusion proteins contain the Fg-binding B-repeat region of M5.</p><p>(D) Schematic representation of the N-terminal region of different fusion proteins. The sequence of the N-terminal region of M22 is given at the top. Asterisks indicate the position of residues L28, E31, and D40 in M22 (corresponding to the three boxed residues in [B]). The ability of the fusion proteins to bind C4BP, indicated to the right, is based on the results shown in (E).</p><p>(E) Ability of fusion proteins to bind C4BP. The fusion proteins (D) are referred to as M22⁵⁷–M5, etc. Whole-cell lysates of E. coli strains, expressing the indicated proteins from genes carried on pBR322, were analyzed by Western blot using Fg or C4BP as the probe. The strain expressing M5 was used as a negative control. The control blot with Fg showed that the proteins were expressed in <i>E. coli.</i> The presence of double bands probably reflects incomplete processing of signal peptides in E. coli and/or intracellular degradation of M protein in this heterologous host.</p></div

DataSheet1_Comparing α-Synuclein Fibrils Formed in the Absence and Presence of a Model Lipid Membrane: A Small and Wide-Angle X-Ray Scattering Study.pdf

Amyloid fibrils are associated with a number of different neurodegenerative diseases. Detailed knowledge of the fibril structure will be of importance in the search of therapy and may guide experiments to understand amyloid formation. In this paper we investigate the morphology of α-synuclein amyloid fibrils, associated with Parkinson’s disease, formed under different conditions. In particular, we study, by means of small and wide-angle X-ray scattering, whether the presence of model lipid membranes affect the overall structure of the fibrils formed, motivated by the fact that amyloid fibrils in vivo are formed in a highly lipid-rich environment. Comparing fibrils formed in the presence of lipid with fibrils formed in their absence, show that the presence of lipids has no detectable effect on the fibril cross-section radius and that the characteristic β-strand repeat distance of 4.7 Å of the extended intermolecular β-sheets remains unaffected. We also show that the observed fibril radius is consistent with a fibril structure composed of two protofilaments. This indicates overall that the particular fibril structure, with their stacks of two-dimensionally folded α-synuclein molecules, represent a deep free energy minimum, not largely affected by the co-aggregation with lipids.</p