Protein Aggregation Kinetics Research Articles

Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.

Kinetics of Protein Aggregation with Formation of Unreactive Intermediates

Irreversible protein aggregation resulting in formation and deposition of insoluble fibrils or amorphous precipitates is usually assumed to occur via sequential attachment of monomers to soluble intermediates. We complement this scheme by slow conversion of the intermediates to a relatively stable form so that they do not react with monomers but can be trapped by precipitates. For reasonable values of parameters, our model predicts that the aggregation kinetics order may be between 2.0 and 2.5. In particular, the model can be used to explain the reaction order, 2.17 +/- 0.09, observed for aggregation of recombinant human granulocyte colony stimulating factor.

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

Effect of protein–protein interactions on protein aggregation kinetics

The mutual diffusion coefficient Dm of dilute lysozyme solutions is measured by the dynamic light scattering technique. The relation between the osmotic second virial coefficient B22 and the protein mutual diffusion coefficient Dm of dilute solutions is examined experimentally and theoretically. The role of B22 in relation with Dm in protein aggregation kinetics was analyzed based on the effect of the average protein pairwise interactions on the protein aggregation kinetics. The empirical “crystallization window” proposed by George and Wilson [Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 361 (1994)] can be interpreted from the point view of crystallization kink kinetics.

Rheological investigation of swollen gluten polymer networks: effects of process parameters on cross‐link density

AbstractWheat gluten samples were subjected to different thermo‐mechanical treatments. Kinetics of protein aggregation and changes in network structure were investigated through biochemical and rheological measurements. Temperature induced protein aggregation through disulphide cross‐links. Shear treatment alters the aggregation mechanism since a lower energy of activation was observed. Accumulation of aggregated protein enhances the elastic behaviour of the material. The strong correlation found between the extent of protein aggregation and the molecular weight between cross‐links reveals the important role of covalent bonds in the network connectivity.

Kinetics of heat- and acidification-induced aggregation of firefly luciferase

The general approach to analysis of the kinetics of protein aggregation registered by the turbidimetric method has been elaborated. The terminal part of the kinetic curves is analyzed using a theoretical equation connecting the derivative of the apparent absorbance ( A) with respect to time (d A/d t) and A ( t is time). This analysis allows the limiting value of A at t→∞ ( A lim) and the order of aggregation with respect to protein ( n) to be calculated. Approach proposed was applied to analysis of thermal and acidification-induced aggregation of firefly luciferase. In both cases the A lim value is a linear function of the protein concentration. The terminal part of the kinetic curves of thermal aggregation follows the first-order kinetics ( n=1), whereas the kinetics of acidification-induced aggregation are characterized by the value of n higher than unity ( n=1.29). The mechanism of nucleation-dependent aggregation has been discussed.

Effects of amylopectin structure and molecular weight on microstructural and rheological properties of mixed beta-lactoglobulin gels.

Nongelling amylopectin fractions from potato and barley have been used to form mixed beta-lactoglobulin gels. The amylopectin fractions were produced by varying the time of alpha-amylase hydrolysis followed by sequential ethanol precipitation. The molecular weights, radius of gyration, chain length distribution, and viscosity of the fractions were established. The mixed gels were analyzed rheologically with dynamic mechanical analysis in shear and microstructurally with light microscopy, transmission electron microscopy, and nuclear magnetic resonance spectroscopy. The result of the gel studies clearly showed that small differences in the molecular weight of amylopectins have a significant influence on the kinetics of protein aggregation and thereby on the gel microstructure and the rheological behavior of the gel. Both an increase in the molecular weight and a higher concentration of amylopectins resulted in a more open protein network structure, with thicker strands of larger and more close-packed beta-lactoglobulin clusters, which showed a larger storage modulus. The transmission electron micrographs revealed that degraded amylopectins were enclosed inside the protein clusters in the mixed gels, whereas nondegraded amylopectin was only found outside the protein clusters. The volume-weighted mean value of the molecular weight of the amylopectins was found to vary between 3.2 x 10(4) and 5.0 x 10(7) Da and the ratio of gyration between 14 and 61 nm. The maximum in chain length distribution was generally somewhat distributed toward longer chain lengths for potato compared to barley, but the differences in chain length distribution were minor compared to those seen in the molecular weight and ratio of gyration between the fractions.

Irreversible aggregation of recombinant bovine granulocyte-colony stimulating factor (bG-CSF) and implications for predicting protein shelf life

The kinetics of irreversible aggregation of bovine Granulocyte-Colony Stimulating Factor (bG-CSF) in solution were investigated as a function of temperature (T), concentration, and pH, and analyzed in terms of an Extended Lumry-Eyring model of protein aggregation proceeding via a non-native conformational state. In the spirit of classic Lumry-Eyring models, the observed kinetics are separated into contributions from thermodynamic or conformational stability of unaggregated native and non-native states, and the intrinsic aggregation kinetics of non-native molecules. It is found that a detailed treatment of the intrinsic kinetics coupled with a two-state approximation of the reversible unfolding transition is sufficient to allow quantitative prediction of low-T stability from high-T data despite highly non-Arrhenius kinetics. Accounting for shifts in conformational equilibrium quantitatively captures the non-Arrhenius T dependence, without requiring the assumption of a change in the rate-determining step with T. From a more general perspective, the observed aggregation behavior of bG-CSF is consistent with the rate-determining step being aggregation at T below a crossover temperature Tx that is inversely related to initial protein concentration. Above Tx, irreversible unfolding is presumably the rate-determining step. The results illustrate that protein aggregation kinetics can, in principle, be predicted quantitatively from so-called accelerated data provided the thermodynamic and kinetic components can be separately extrapolated to longer term storage conditions.

Impact of glycerol on thermostability and heat-induced gelation of bovine serum albumin

The influence of glycerol (0–40 wt%) on the thermal denaturation and gelation of bovine serum albumin (BSA) in aqueous solution has been studied. The effect of glycerol on heat denaturation of 0.5 wt% BSA solutions (pH 7.0) was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and was characterized by a denaturation temperature ( T m). There was no significant change in T m as the glycerol concentration was increased from 0 to 40 wt%, suggesting that glycerol interacted similarly with the native and heat-denatured forms of the protein. The dynamic shear rheology of 4 wt% BSA solutions containing 200 mM NaCl was monitored as they were heated from 30 to 90 °C at 1.5 °C min −1, held at 90 °C for 70 min, and then cooled back to 30 °C at −1.5 °C min −1. The turbidity of the same solutions was monitored as they were heated from 30 to 90 °C at 1.5 °C min −1 or held isothermally at 90 °C for 10 min. Glycerol increased the protein gelation temperature ( ΔT gel ∼+3 ° C for 40 wt% glycerol) and decreased the gelation rate, which was attributed primarily to its ability to decrease the protein–protein collision frequency by increasing solution viscosity. The presence of glycerol either increased or decreased the shear modulus of the gels depending on its concentration and temperature, which was attributed to its ability to alter protein aggregation kinetics during gelation and its ability to increase the attractive forces between proteins at lower temperatures.

Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation.

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A(lim) and kI (A(lim) is the limiting value of A at t --> infinity and kI is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant kI calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A(lim) and kI may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.

17] Analysis of protein aggregation kinetics

Given a set of kinetic data, then, the preceding discussions suggest the following approach to its analysis. 1. For purposes of establishing the reaction, ignore the final stages and concentrate on the initial 10-20% of the reaction at first. A globally optimized model may be based on a faulty assumption for the initial steps. Thus, although the whole data set may look reasonably well fit, the reaction could be misrepresented, and thus the fit unhelpful if accuracy at the later stages has come at the expense of the initial phase of the reaction. 2. What is the time course of the initial reaction? (A) Is the reaction exponential? Exponential growth gives dramatic lag times (see Fig. 3), whereas nonexponential "lag times" have a visible signal from time 0 (i.e., Fig. 2). If the data set shows the abrupt appearance of signals after a period of quiescence, the chances are excellent that the time course is exponential. High sensitivity measurement of the signal at times during the lag phase should be used to confirm the exponential nature quantitatively. Exponential reactions mean a secondary pathway is operative. (a) A cascade (tn) can look similar to an exponential, but may proceed from a multistep single-path reaction. Thus the exponential needs to be ascertained with some accuracy. (b) It is possible that some or all of the lag results from a stochastic process, i.e., formation of a single nucleus being observed. This, however, is likely to be accompanied by a secondary process, as few techniques are sensitive enough to detect a single polymer at a time, and having one nucleus form many polymers is a hallmark of a secondary process. Thus, the reproducibility of the kinetics must be established to rule out stochastics. If data show wide variation, stochastic methods as described earlier may be employed. (c) Given a secondary process, one must separate the primary nucleation process from the secondary process (by stochastic means or by use of the product B2A, as described earlier). (B) If the reaction does not begin with an exponential, is it parabolic? If so, it falls in the general class of linear polymerizations. 3. What is the concentration dependence of the reaction(s)? This will separate nucleation processes from growth, and so on. 4. If the initial reaction is neither exponential nor parabolic, a reaction mechanism needs to be proposed and evaluated. Solving the resulting equations is best done by linearization, which has the best chance of giving equations whose solutions and their sensitivity to parameters are readily understood. If this proves fruitful, full numeric solutions may be useful. 5. At this point, the full reaction may be considered to completion. 6. The physical basis of the description (sizes of parameters and their dependencies) needs to be finally considered to ensure that the mathematical success of the description rests on tenable physical grounds.

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.

Thermal Stability and Functionality of Whey Proteins

Functional properties of whey proteins are often impaired by inevitable heat treatments during processing of whey protein products and preservation of food products. The extent of deterioration depends on the relation between the rate of protein denaturation and the heat load applied. In a new kinetic approach, whey protein denaturation is split into two successive processes: 1) unfolding, determined by differential scanning calorimetry; and 2) aggregation, derived from high performance gel permeation chromatography.A short review is presented of industrial recovery processes of whey protein products and their related functional properties. The effect of heat treatments between 70 and 90°C is analyzed in terms of the kinetics of whey protein unfolding and aggregation. The results reveal that thermal denaturation of whey proteins during industrial heating processes is predictable. An important determinant for functional properties is the salt composition of whey protein products during heat treatments at temperatures above 75°C.

Aggregation kinetics in salt‐induced protein precipitation

AbstractThe kinetics of protein aggregation in salt‐induced precipitation processes were studied as a function of salt type, salt concentration, temperature and protein concentration. α‐chymotrypsin (αCT) was used as a model protein. Stopped‐flow turbidimetry was used to monitor the progress of precipitations. Analysis of the linear portions of the turbidity trajectories indicates that temperature and salt concentration effects are related to protein solubility; the protein concentration dependence is well described by the Smoluchowski collision equation. The aggregation kinetics of partially‐inhibited αCT exhibit poisoning behavior, underscoring the importance of dimerization and monomer addition in the precipitation of αCT. Solute particle radius distributions determined via dynamic laser light scattering for low salt and supernatant αCT solutions indicated that significant aggregation does not occur in the absence of supersaturation. A detailed population balance model was proposed that accounted for specific and nonspecific interactions and monomer addition. The model is expected to find general application to protein aggregation phenomena, in particular for proteins that have specific quaternary interactions.

High-performance liquid chromatography of amino acids, peptides and proteins : LXXXVI. The influence of different displacer salts on the retention and bandwidth properties of proteins separated by isocratic anion-exchange chromatography

The influence of different displacer salts on the retention behaviour of seven globular proteins ranging in moleclar weight from 12 000 to 69 000 was investigated using the Mono Q anion-exchange resin as the stationary phase. Isocratic retention data were collected using several different alkali metal halides as the displacing salt, thereby systematically varying the anion and cation species in the series F −, Cl − and Br − and Li +, Na + and K +. The different anions were found to reduce protein retention in order of their decreasing hydrated ionic radii. Protein Z c values were found to be lower for fluoride and bromide than for chloride. It was demonstrated that the cationic co-ions also influence solute retention properties with this anion-exchange resin through, inter alia, preferential interactions with the protein solute. Protein band-broadening was found to systematically vary with the choice of displacer salt. These changes were related to known Hofmeister effects on protein aggregation kinetics and solubility and the degree of ion penetration at the double layer of the stationary phase-mobile phase interface. These studies now provide a rapid comparative basis for evaluating the mechanism of co- and counter-ion interactions with proteins in high-performance ion-exchange chromatographic systems.

Electrostatic interaction and the kinetics of protein aggregation: αs1-casein

Detailed studies have been made of the rates of the calcium-ion-induced precipitation of bovine α s 1 -casein under known conditions of calcium-ion binding. From these data it has been found possible to explain the precipitation as being caused by the reduction in the negative charge of the protein resulting from the binding of (positive) calcium ions. Simple Coulombic theory predicts that the logarithm of the rate constant for the interaction of charged protein particles is dependent on the square of their charge, and this relationship was found to apply to the precipitating calcium-caseinate complexes. Consideration of the reaction in terms of more realistic theories of colloid chemistry also predicts the observed charge dependence. It is concluded that the precipitation of the Ca 2+- α s 1 -caseinate complex is governed by an energy barrier whose height is dependent on the overall charge of the complex, and that the precipitation is compatible with theories of the stability of lyophobic colloids.

A simple model of the reaction between polyadenylic acid and polyuridylic acid

Protein aggregation is an important phenomenon that alternatively is part of the normal functioning of nature or, central to this review, has negative consequences via its hypothesized central role in neurodegenerative diseases. A key to controlling protein aggregation is understanding the mechanism(s) of protein aggregation. Kinetic studies, data curve-fitting, and analysis are, in turn, keys to rigorous mechanistic studies. The main goal of this review is to analyze and report on the primary literature contributions to protein aggregation kinetics, mechanism, and curve-fitting. Following a brief introduction, the multiple different physical methods that have been employed to follow protein aggregation are presented and briefly discussed. Next, key information on the starting proteins and especially the products, and any detectable intermediates, involved in protein aggregation are presented. This is followed by tabulation (in the Supporting information) and discussion (in the main text), of the many approaches in the literature striving to determine the kinetics and mechanism of protein aggregation. It is found that these approaches can be broadly divided into three categories: (i) kinetic and thermodynamic, (ii) empirical, and (iii) other approaches. The first two approaches are the main focus of the present contribution, their goal being curve-fitting the available kinetic data and obtaining quantitative rate constants characterizing the nucleation, growth, and any other parts of the overall aggregation process. The large literature of protein aggregation is distilled down to five classes of postulated mechanisms: i) the subsequent monomer addition mechanism, ii) the reversible association mechanism, iii) prion aggregation mechanisms, iv) an “Ockham's razor”/minimalistic model first presented in 1997 and known as the Finke–Watzky 2-step model, and v) quantitative structure activity relationship models. These five classes of mechanisms are reviewed in detail in historical order; where possible corresponding kinetic equations, and fits to aggregation data via the proposed mechanisms, are analyzed and discussed. The five classes of mechanisms are then analyzed and discussed in terms of their similarities and differences to one another. Also included is a brief discussion of selected empirical approaches used to investigate protein aggregation. Three problem areas in the protein aggregation kinetic and mechanistic studies area are identified, and a Summary and Conclusions section is provided en route to moving the field forward towards the still unachieved goal of unequivocal elucidation of the mechanism(s) of protein aggregation.