Prion proteins: folding and aggregation properties

The partial unfolding or alternative folding of a class of polypeptides is at the origin of fascinating events in living cells. In their non-native conformation, these constitutive polypeptides called prions are at the origin of a protein-based structural heredity. These polypeptides are closely associated to a class of fatal neurodegenerative illnesses in mammals and to the emergence and propagation of phenotypic traits in baker's yeasts. The structural transition from the correctly folded, native form of a prion protein to a persistent misfolded form that ultimately may cause cell death or the transmission of phenotypic traits is not yet fully understood. The mechanistic models accounting for this structure-based mode of inheritance and the extent of partial unfolding of prions or their alternative folding and the subsequent aggregation process are developed and discussed. Finally, the potential regulation of prion propagation by molecular chaperones is presented.     

Gating and conduction of nano-channel forming proteins: a computational approach

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane β-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel’s structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:3     

Different circular permutations produced different folding nuclei in proteins: a computational study

There have been many studies about the effect of circular permutation on the transition state/folding nucleus of proteins, with sometimes conflicting conclusions from different proteins and permutations. To clarify this important issue, we have studied two circular permutations of a lattice protein model with side-chains. Both permuted sequences have essentially the same native state as the original (wild-type) sequence. Circular permutant 1 cuts at the folding nucleus of the wild-type sequence. As a result, the permutant has a drastically different nucleus and folds more slowly than wild-type. In contrast, circular permutant 2 involves an incision at a site unstructured in the wild-type transition state, and the wild-type nucleus is largely retained in the permutant. In addition, permutant 2 displays both two-state and multi-state folding, with a native-like intermediate state occasionally populated. Neither the wild-type nor permutant 1 has a similar intermediate, and both fold in an apparently two-state manner. Surprisingly, permutant 2 folds at a rate identical with that of the wild-type. The intermediate in permutant 2 is stabilised by native and non-native interactions, and cannot be classified simply as on or off-pathway. So we advise caution in attributing experimental data to on or off-pathway intermediates. Finally, our work illuminates the results on alpha-spectrin SH3, chymotrypsin inhibitor 2 and beta-lactoglobulin, and supports a key assumption in the experimental efforts to locate potential nucleation sites of real proteins via circular permutations.     

Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins

Hydrophobicity is thought to be one of the primary forces driving the folding of proteins. On average, hydrophobic residues occur preferentially in the core, whereas polar residues tend to occur at the surface of a folded protein. By analyzing the known protein structures, we quantify the degree to which the hydrophobicity sequence of a protein correlates with its pattern of surface exposure. We have assessed the statistical significance of this correlation for several hydrophobicity scales in the literature, and find that the computed correlations are significant but far from optimal. We show that this less than optimal correlation arises primarily from the large degree of mutations that naturally occurring proteins can tolerate. Lesser effects are due in part to forces other than hydrophobicity, and we quantify this by analyzing the surface-exposure distributions of all amino acids. Lastly, we show that our database findings are consistent with those found from an off-lattice hydrophobic-polar model of protein folding.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Local sequence patterns of hydrophobicity and solvent accessibility in soluble globular proteins

We examined the variation in the solvent accessibility and hydrophobicity of the amino acids along the sequences of 58 soluble globular proteins with known tertiary structure. We found that there is a significant tendency for the accessibilities to run in clusters along the sequence but that the hydrophobicities are distributed without such nonrandom clusters. Theseresults suggest severe limitations on the power of sequence analysis tools that use average hydrophobicity scores of overlapping subsequences to predict accessibility.     

Derivation of a solubility condition for proteins from an analysis of the competition between folding and aggregation

Failure in maintaining protein solubility in vivo impairs protein homeostasis and results in protein misfolding and aggregation, which are often associated with severe neurodegenerative and systemic disorders that include Alzheimer's and Parkinson's diseases and type II diabetes. In this work we formulate a model of the competition between folding and aggregation, and derive a condition on the solubility of proteins in terms of the stability of their folded states, their aggregation propensities and their degradation rates. From our model, the bistability between folding and aggregation emerges as an intrinsic aspect of protein homeostasis. The analysis of the conditions that determine such a bistability provides a rationalization of the recently observed relationship between the cellular abundance and the aggregation propensity of proteins. We then discuss how the solubility condition that we derive can help rationalise the correlation that has been reported between evolutionary rates and expression levels or proteins, as well as in vivo protein solubility and expression level measurements, and recently elucidated trends of proteome evolution.     

Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach

Journal of Molecular Modeling - In order to predict the impact sensitivity of high explosives, we designed and evaluated several models based on the trigger linkage hypothesis and the Arrhenius...     

Rattling the cage: computational models of chaperonin-mediated protein folding

Chaperonins are known to maintain the stability of the proteome by facilitating the productive folding of numerous misfolded or aggregation-prone proteins and are thus essential for cell viability. Despite their established importance, the mechanism by which chaperonins facilitate protein folding remains unknown. Computer simulation techniques are now being employed to complement experimental ones in order to shed light on this mystery. Here we review previous computational models of chaperonin-mediated protein folding in the context of the two main hypotheses for chaperonin function: iterative annealing and landscape modulation. We then discuss new results pointing to the importance of solvent (a previously neglected factor) in chaperonin activity. We conclude with our views on the future role of simulation in studying chaperonin activity as well as protein folding in other biologically relevant confined contexts.     

The folding mechanism of a beta-sheet: the WW domain

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel beta-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Phi(m)) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 degrees C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi(T), obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi(m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi(T) to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.     

Protein folding and aggregation: two sides of the same coin in the condensation of proteins revealed by pressure studies

Hydrostatic pressure can be considered as "thermodynamic tweezers" to approach the protein folding problem and to study the cases when folding goes wrong leading to the protein folding disorders. The main outcome of the use of high pressure in this field is the stabilization of folding intermediates such as partially folded conformations, thus allowing us to characterize their structural properties. Because partially folded intermediates are usually at the intersection between productive and off-pathway folding, they may give rise to misfolded proteins, aggregates and amyloids that are involved in many neurodegenerative diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease and Huntington's disease. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform, the PrP(Sc) (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. The last findings on the application of high pressure on amyloidogenic proteins will be discussed here as regards to their energetic and volumetric properties. The use of high pressure promises to contribute to the identification of the underlying mechanisms of these neurodegenerative diseases and to develop new therapeutic approaches.     

Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins

Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteions.     

The role of electrostatics in TrxR electron transfer mechanism: A computational approach

Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a “shuttle,” the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson–Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N‐terminal (FAD and Cys59/64) and C‐terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired “shuttle.” Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the “shuttle” mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836–1843. © 2016 Wiley Periodicals, Inc.     

Analysing the patterning of a sequence of discrete behavioural events

Existing techniques for analysing the organization of behavioural events are discussed. Cluster analysis, mutual replaceability, ‘melody’ detection and Markov analyses are examined in order to discover the validity and usefulness of the concepts searched for by each technique, and the mathematical and statistical background of the techniques is scrutinized. Some new techniques, designed to search for and detect possible important sequential organization patterns, are described in the light of the previous criticisms. Of these techniques, the Pre-post-state Histogram (PPSH)—a multi-order Markov graphical analysis—would appear to be the most important for future work, especially when used in conjunction with the other techniques described.     

Extinction risk of a meta-population: aggregation approach

Aggregation of variables of a complex mathematical model with realistic structure gives a simplified model which is more suitable than the original one when the amount of data for parameter estimation is limited. Here we explore use of a formula derived for a single unstructured population (canonical model) in predicting the extinction time for a population living in multiple habitats. In particular we focus multiple populations each following logistic growth with demographic and environmental stochasticities, and examine how the mean extinction time depends on the migration and environmental correlation. When migration rate and/or environmental correlation are very large or very small, we may express the mean extinction time exactly using the formula with properly modified parameters. When parameters are of intermediate magnitude, we generate a Monte Carlo time series of the population size for the realistic structured model, estimate the "effective parameters" by fitting the time series to the canonical model, and then calculate the mean extinction time using the formula for a single population. The mean extinction time predicted by the formula was close to those obtained from direct computer simulation of structured models. We conclude that the formula for an unstructured single-population model has good approximation capability and can be applicable in estimating the extinction risk of the structured meta-population model for a limited data set.