The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering
Introduction
Amorphous (unordered) aggregates of different proteins have been recently implicated in pathogenesis of many important human diseases [1], [2], [3], [4], [5]. However, structural studies of such aggregates are greatly hampered by their large size, transient character, and heterogeneity. Probably, because of this, up to now, as far as we know, only one detailed model of mechanism of amorphous protein aggregation has been proposed. This is the Goldberg–Wetzel model [6], [7] according to which unordered aggregates are formed by intermolecular interactions of those domains of partly disordered protein molecules, which in the native state were involved in intramolecular interactions between the same domains.
Tobacco mosaic virus (TMV) coat protein (CP) is well known for its ability to produce ordered assemblies [8]. It is well known that at room temperature, pH values in the range from 7.5 to 9.0, and ionic strength in the range from 10 to 100 mM, the TMV CP exists in the form of so called 4S-protein (a dynamic mixture of pentamers and trimers, with a minor amount of monomers). At pH of about 7.0 and ionic strength of about 100 mM TMV CP with high efficiency specifically assembles in vitro with TMV RNA with formation of completely native virions. At pH ≤ 6.0 in the absence of RNA, the protein produces long virus-like helical aggregates called repolymerized protein [8].
However, TMV CP also turned out to be a good model for studies of thermal amorphous aggregation. In contrast to most of other unspecific protein aggregation system [7], [9], the process of amorphous TMV CP aggregation is highly reproducible and its rate can be easily manipulated by changing solution ionic strength, protein concentration, and temperature [10], [11], [12], [13]. Recently we have observed that the TMV CP amorphous aggregation can be also induced in neutral phosphate buffer (PB) at room temperature (25 °C) by low micromolar concentrations of cationic surfactant cetyltrimethylammonium bromide (CTAB) [14].
And finally, possible existence of amyloidogenic potential in TMV CP have been suggested by R. Diaz-Avalos and D. Caspar from the results of their studies of the TMV CP “off-pathway” aggregate (stacked disks) structure [15].
Dynamic light scattering (DLS) gives valuable information on the size of protein aggregates and is widely used for the study of the kinetics of protein aggregation. DLS, as applied to analysis of protein aggregates, has some evident merits. Modern variants of DLS technique allow registering the initial stages of protein aggregation, where the fraction of the aggregated protein constitutes some tenth of a percent of the total amount of the protein in the system. Besides, this method allows quantitative determination of the individual components in heterogeneous populations of aggregates [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26].
Recently we have studied with the help of DLS the process of aggregation of βL-crystallin in the presence of α-crystallin. We have found that three different types of amorphous aggregates (named “start aggregates”, “basic aggregates” and “superaggregates”) are formed [27]. The construction of the light scattering intensity versus hydrodynamic radius plot allows estimating the size of the start aggregates [27]. The formation of the start aggregates (the primary clusters) has been also observed on thermal aggregation of several other proteins [27], [28], [29].
The goal of the present work was to carry out the comparative study of the kinetics of irreversible heat-induced and reversible CTAB-induced amorphous aggregation of the TMV CP by DLS. It has been shown that in both cases aggregation proceeds by way of the formation of the start aggregates.
Section snippets
TMV purification and proteins preparation
Wild-type (strain U1) TMV was obtained as described elsewhere [30] and its coat protein was isolated by the acetic acid method [31]. The CP preparations were stored at concentrations of 4.5 to 6 mg/ml (259–345 μM) in 5 mM Na+/Na+ PB, pH 8.0, at + 4 or − 20 °C. The TMV CP concentrations were measured by UV spectroscopy using the extinction coefficient A2800.1% of 1.30 [32].
α-Crystallin from bovine eye lenses was purchased from Sigma (USA).
DLS measurements
DLS is widely used in biochemistry to measure the size of
The kinetics of thermal aggregation of TMV CP
Dynamic light scattering allows the changes in the size of aggregates in the course of protein aggregation to be registered. Fig. 1 shows the typical autocorrelation functions measured at various times of incubation of 5.75 μM (0.1 mg/ml) TMV CP in 50 mM PB at 52 °C. Using the DynaLS software we calculated the size distribution of particles formed in the course of aggregation. At sufficiently short times of incubation the distribution function was unimodal (Fig. 2A, B). The mean value of the
Discussion
With the help of DLS it was found that two types of aggregates (basic aggregates and superaggregates) are formed in the course of the TMV CP thermal aggregation (Fig. 2, Fig. 3). The splitting of aggregate population into two components is not a general phenomenon, but it was registered by DLS for thermal aggregation of bovine serum albumin at 58 °C [26], aggregation of dithiothreitol-denatured α-lactalbumin [43], and thermal aggregation of βL-crystallin in the presence of α-crystallin [27].
Conclusions
The results of the present and previous [27], [28], [29] studies testify that in many cases amorphous protein aggregation begins with the formation of rather large start aggregates (with the Rh value from 20 to 100 nm) and further occurs according to the diffusion-limited cluster–cluster aggregation mechanism [35], [36], [37], [38]. Nevertheless, important differences in the process of aggregation are observed for individual systems. In the course of thermal aggregation of mixtures of βL- and
Acknowledgements
This work was supported by the Russian Foundation for Basic Research (grants 05-04-49503 and 05-04-48691), the Program for Fundamental Research “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences, and INTAS (grant 03-51-4813).
References (43)
Mutations and off-pathway aggregation of proteins
Trends Biotechnol.
(1994)Aggresomes, inclusion bodies and protein aggregation
Trends Cell Biol.
(2000)- et al.
A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein
Int. J. Biochem. Cell Biol.
(2003) - et al.
Low cetyltrimethylammonium bromide concentrations induce reversible aggregation of tobacco mosaic virus and it's coat protein at room temperature
Int. J. Biochem. Cell Biol.
(2006) - et al.
Hyperstable stacked-disk structure of tobacco mosaic virus protein: electron cryomicroscopy image reconstruction related to atomic models
J. Mol. Biol.
(2000) - et al.
Heat-induced aggregation of beta-lactoglobulin studied by dynamic light scattering
Int. Dairy J.
(1996) - et al.
Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus
Biophys. J.
(1996) - et al.
Thermally induced aggregation of human transferrin receptor studied by light-scattering techniques
Biophys. J.
(1999) - et al.
Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small shock superfamily
Biophys. J.
(2001) - et al.
Structure of aggregating kappa-carrageenan factor studied by light scattering
Int. J. Biol. Macromol.
(2001)
Kinetics of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin
Int. J. Biol. Macromol.
Assembly of amyloid protofibrils via critical oligomers — a novel pathway of amyloid formation
J. Mol. Biol.
Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering
Biophys. Chem.
Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering
Biophys. Chemist.
Degradation of tobacco mosaic virus with acetic acid
Virology
Structure of single-stranded virus RNA in situ. II. Optical activity of five tobacco mosaic-like viruses and their components
Biochem. Biophys. Acta
Alpha-crystallin as a molecular chaperone
Prog. Retin. Eye Res.
The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin
Biochem. Biophys. Res. Commun.
Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases
Nature
Spherical aggregates of beta-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3beta
Proc. Natl. Acad. Sci. U. S. A.
Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis
Science
Cited by (37)
Glucose-induced structural changes and anomalous diffusion of elastin
2020, Colloids and Surfaces B: BiointerfacesProtein folding, misfolding and aggregation: A tale of constructive to destructive assembly
2018, International Journal of Biological MacromoleculesPhotodynamic inactivation of bacteriophage MS2: The A-protein is the target of virus inactivation
2018, Journal of Photochemistry and Photobiology B: BiologyCitation Excerpt :DLS measures Brownian motion of particles in solution and correlates this to hydrodynamic radius (DH). The larger the particle, the slower the Brownian motion will be [50]. The peak shifts (Fig. 6B) correspond to increase of average MS2 particle size (d. nm) as the time of PDI increases (Fig. 6C).
A change in the pathway of dithiothreitol-induced aggregation of bovine serum albumin in the presence of polyamines and arginine
2017, International Journal of Biological MacromoleculesQuantification of anti-aggregation activity of chaperones
2017, International Journal of Biological MacromoleculesCitation Excerpt :These approaches can be used for elucidation of the mechanisms of the protective action of chaperones and for measurement of the combined effects of chaperones. When studying thermal aggregation of GAPDH, Phb and creatine kinase from rabbit skeletal muscles, βL-crystallin, tobacco mosaic virus coat protein, yeast alcohol dehydrogenase and mitochondrial aspartate aminotransferase using dynamic light scattering, it was shown that the initial stage of protein aggregation is the formation of start aggregates involving hundreds of the denatured protein molecules [41,43–45,48–58]. The intermediate states between the native protein forms and start aggregates were not detected.