Evidence for the formation of start aggregates as an initial stage of protein aggregation

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3-phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1-3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large-sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity.     

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin

The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule.     

Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.     

Mechanism of chaperone-like activity. Suppression of thermal aggregation of betaL-crystallin by alpha-crystallin

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.     

Effect of alpha-crystallin on thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed.     

Low cetyltrimethylammonium bromide concentrations induce reversible amorphous aggregation of tobacco mosaic virus and its coat protein at room temperature

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.     

Arginine controls heat-induced cluster-cluster aggregation of lysozyme at around the isoelectric point

The process of protein aggregation has attracted a great deal of research attention, as aggregates are first of all a nuisance to preparation of high quality protein and secondly used as novel materials. In the latter case, the process of protein aggregation needs to be controlled. Here, we show how arginine (Arg) regulates the process of heat-induced protein aggregation. Dynamic light scattering and transmission electron microscopy revealed that heat-induced aggregation of lysozyme at around the isoelectric point occurred in a two-step process: formation of start aggregates, followed by further growth mediated by their sticking with diffusion-limited cluster-cluster aggregation. In the presence of Arg, the diffusion-limited regime changed to reaction-limited cluster-cluster aggregation. The data indicated that the solution additives that coexisted with proteins would affect the property of the formed product, such as morphology and mechanic strength.     

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.     

Effect of α-crystallin on thermostability of mitochondrial aspartate aminotransferase

Effect of α-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of α-crystallin. According to the data of differential scanning calorimetry, α-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 °C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of α-crystallin proceeds in the regime of reaction-limited cluster–cluster aggregation.     

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag‐time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size‐exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.     

Comparative Analysis of the Effects of α-Crystallin and GroEL on the Kinetics of Thermal Aggregation of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase

Effects of α-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of α-crystallin or GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster–cluster aggregation to the regime of reaction-limited cluster–cluster aggregation, wherein the sticking probability for the colliding particles becomes lower the unity. In contrast to α-crystallin, GroEL does not interfere with formation of the start aggregates which include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that the products of dissociation of GAPDH and α-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones at elevated temperatures.     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.     

Effects of small heat shock proteins on the thermal denaturation and aggregation of F-actin

Effect of recombinant chicken small heat shock protein with molecular mass 24 kDa (Hsp24) and recombinant human small heat shock protein with molecular mass 27 kDa (Hsp27) on the heat-induced denaturation and aggregation of skeletal F-actin was analyzed by means of differential scanning calorimetry and light scattering. All small heat shock proteins did not affect thermal unfolding of F-actin measured by differential scanning calorimetry, but effectively prevented aggregation of thermally denatured actin. Small heat shock protein formed stable complexes with denatured (but not with intact) F-actin. The size of these highly soluble complexes was smaller than the size of intact F-actin filaments. It is supposed that protective effect of small heat shock proteins on the cytoskeleton is at least partly due to prevention of aggregation of denatured actin.     

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. I: Biotinylated liposomes and avidin

The aggregation of biotin-modified phospholipid vesicles (liposomes) induced by binding the protein avidin in solution is analyzed experimentally and theoretically. Avidin has four binding sites that can recognize biotin specifically, and is able to cross-link the liposomes to form large aggregates. The aggregation kinetics were followed using quasi-elastic light scattering (QLS) to measure the mean particle size, and by measuring the solution turbidity. The rate and extent of aggregation were determined as a function of vesicle concentration, protein concentration, and the biotin density on the surface of the liposomes. A model based on Smoluchowski kinetics, fractal concepts, and Rayleigh and Mie light scattering theory was developed to analyze the experimental observations. Small aggregates (<7800 A diameter) may be treated as globular; however, the fractal nature of larger particles must be taken into account. Parameters in the model are taken from molecular simulations, or fit to the experimental observations. The aggregation kinetics are primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration. Good agreement is found between the model and the experimental results. (c) 1996 John Wiley & Sons, Inc.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Time resolved structure analysis of growing beta-amyloid fibers

Formation of beta-amyloid plaques is a crucial feature of Alzheimer's disease. In the present work time resolved static light scattering was applied to investigate the size and shape of growing beta-amyloid aggregates preceding plaque formation. The beta-amyloid protein with 40 amino acid residues was used. Salt free buffer solutions and solutions with 0.15M NaCl at 37 degrees C served as the aggregation medium. The focus lay on the first 2h following initiation of the aggregation process which corresponds to the protofibril phase. Addition of the NaCl accelerated the aggregation process considerably. Scattering data from aggregation in saline solutions indicated formation of long fibers which suggest interpretation of data with the worm-like chain model. Two important results were revealed: (i) At the end of the time resolved recordings, the worm-like chain model provided a fully adequate picture for the growing aggregates. Chain stiffness is characterised in terms of the persistence length, which is close to 50 nm. The linear mass density of the growing fibers approached a value of two monomers per nm corresponding to single stranded fibers, which is in accordance with presently existing models for the aggregation of beta-amyloid. The fibers finally reached contour lengths of several thousand nanometers. (ii) The plateau values for the persistence length and linear mass density observed in the final regime are gradually approached from higher values. This observation is inconsistent with simple worm-like chains. Rather does it indicate existence of another species during the initial phase of the aggregation, in addition to monomers and fibers. Aside from further insight into fundamental aspects of beta-amyloid aggregation, time resolved static light scattering provides an appropriate tool for assay tests with drugs designed to interfere with the aggregation process.     

Influence of metal ions on thermal aggregation of bovine serum albumin: Aggregation kinetics and structural changes

Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to β-structures to the detriment of α-helix conformation of BSA. The temperature of protein unfolding was not sensitively affected by the presence of Zn(II) or Cu(II) ions; on the contrary, only Zn(II) ions slightly promoted the heat-induced aggregation of the protein, since bigger aggregates were formed in their presence. The different efficacy of the Cu(II) and Zn(II) ions in promoting the BSA aggregation were highlighted by Raman measurements, assessing the role of His residues in metal binding. A distinct polypeptide folding of the two metal-BSA systems takes place, since the predominant mode of metal binding depends on metal. In particular, in Zn-BSA the metal coordination involves the imidazole N_τ atom of His which can promote inter-molecular cross-linking.