Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5.5 to 7.0. Sucrose-density-gradient studies in deuterium oxide at pD6.0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E(620)/E(280) ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.     

Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge

Solutions of C-phycocyanin of very low concentrations were examined by sedimentation-velocity studies in the Spinco model E ultracentrifuge equipped with a photoelectric scanning system and a monochromator. At sufficiently low concentrations complete disaggregation from the hexamer to the monomer was observed. The equilibrium constant of monomer to hexamer was estimated to be approx. 10(30). For studies of aggregation over the complete range of concentration, C-phycocyanins from Phormidium luridum and Lyngbya sp. were used. Sedimentation-velocity studies at high concentration with schlieren optics are reported for C-phycocyanins from Anabaena variabilis and Lyngbya sp. The pH-dependence of aggregation and the temperature-dependence of trimer-hexamer equilibrium for phycocyanins from these algae were found to be similar to those of other C-phycocyanins. The principal feature of the pH-dependence is the dominance of hexamers at the isoelectric point. Increasing temperature increased the amount of hexamer and decreased the amount of trimer.     

Increased aggregation of C-phycocyanin produced by phenol and benzene

The effect of selected aromatic compounds on aggregates of C-phycocyanin was examined by analytic ultracentrifugation. Benzene and phenol caused an increase in aggregation. The principal change, a conversion of 6S to an 11S species, is postulated to result from the interaction of benzene and phenol with hydrophobic areas involved in protein-protein contact.The C-phycocyanin used in these studies included that extracted from Spirulina platensis, a Cyanophyta that grows in a highly alkaline environment. The pH dependence of the aggregation properties of this protein, however, was similar to that of C-phycocyanins from algae grown in normal environments. This suggests that the internal biopolymers of organisms which naturally exist at extremes of pH are somehow protected from exposure to these extremes.     

Protein aggregation in silico

Protein aggregation is a challenge to the successful manufacture of protein therapeutics; it can impose severe limitations on purification yields and compromise formulation stability. Advances in computer power, and the wealth of computational studies pertaining to protein folding, have facilitated the development of molecular simulation as a tool to investigate protein misfolding and aggregation. Here, we highlight the successes of protein aggregation studies carried out in silico, with a particular emphasis on studies related to biotechnology. To conclude, we discuss future prospects for the field, and identify several biotechnology-related problems that would benefit from molecular simulation.     

Protein misfolding and aggregation

Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: (1) the sharp rise in the number and volume of therapeutic proteins produced commercially and (2) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco.     

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.     

Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates

Noncovalent aggregation is a side reaction in the process of reconstitution of oligomeric enzymes (e.g., lactic dehydrogenase) after preceding dissociation, denaturation, and deactivation. The aggregation product is of high molecular weight and composed of monomers which are trapped in a minium of conformational energy different from the one characterizing the native enzyme. This energy minimum is protected by a high activation energy of dissociation such that the aggregates are perfectly stable under nondenaturing conditions, and their degradation is provided only by applying strong denaturants, e.g., 6 M guanidine hydrochloride at neutral or acidic pH. The product of the slow redissolution process is the monomeric enzyme in its random configuration, which may be reactivated by diluting the denaturant under optimum conditions of reconstitution. The yield and the kinetics of reactivation of lactic dehydrogenase from pig skeletal muscle are not affected by the preceding aggregation-degradation cycle and are independent of different modes of aggregate formation (e.g., by renaturation at high enzyme concentration or heat aggregation). The kinetics of reactivation may be described by one single rate-determining bimolecular step with k2 = 3.9 x 10(4) M-1 s-1 at zero guanidine concentration. The reactivated enzyme consists of the native tetramer, characterized by enzymatic and physical properties identical with those observed for the enzyme in its initial native state.     

Characterization of chromophoric peptides from C-phycocyanin.

Three chromophoric peptides have been isolated and characterized from tryptic digests of the α subunit of C-phycocyanin from $\text{Oscillatoria}$,$\text{agardhii}$. The amino acid sequences revealed that one phycocyanobilin was ester bond by a tyrosine residue, and another was most probably attached by a thioether linkage. Structural studies of the third chromophoric peptide gave no evidence of how the phycocyanobilin was attached.     

Imaging individual protein aggregates to follow aggregation and determine the role of aggregates in neurodegenerative disease

Protein aggregates play a key role in the initiation and spreading of neurodegenerative disease but have been difficult to study due to their low abundance and heterogeneity, in both size and structure. Fluorescence based methods capable of detecting and characterising single aggregates have recently been developed and can be used to measure many important aggregate properties, and can be combined with sensitive assays to measure aggregate toxicity. Here we review these methods and discuss recent examples of their application to determine the molecular mechanism of aggregation and the detection of aggregates in cells and cerebrospinal fluid. The further development of these methods and their application to the aggregates present in humans has the potential to solve a major problem in the field and allow the identification of the key toxic species that should be targeted in therapies.     

Unified effects of aggregation reveal larger prey groups take longer to find

Previous work has suggested that larger groups of prey are more conspicuous to predators. However, this ignores that prey populations are finite. As groups get larger they become fewer, hence the encounter rate between predator and prey decreases with prey aggregation. Here, we present a two-dimensional model based on visual angle to unify these encounter and conspicuousness effects of aggregation. With experimental support using three-spined sticklebacks (Gasterosteus aculeatus L.), searching for chironomid larvae, we demonstrate that the increase in visual angle with increasing group size is outweighed by its corresponding decrease as the groups become fewer and thus further away from the searching predator. The net effect is that prey are found with more difficulty when they aggregate, giving an additional anti-predatory benefit to group living rather than a cost.     

Protein aggregation in Huntington's disease

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.     

Protein aggregation in crowded environments

The generic tendency of proteins to aggregate into non-functional, and sometimes cytotoxic, structures poses a universal problem for all types of cell. This tendency is greatly exacerbated by the high total concentration of macromolecules found within most intracellular compartments, a phenomenon referred to as macromolecular crowding. This review discusses the quantitative effects of crowding on protein aggregation and the role of molecular chaperones in combating this problem.     

Protein aggregates stimulate macropinocytosis facilitating their propagation

Temporal and spatial patterns of pathological changes such as loss of neurons and presence of pathological protein aggregates are characteristic of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Frontotemporal Dementia, Alzheimer's disease and Parkinson's disease. These patterns are consistent with the propagation of protein misfolding and aggregation reminiscent of the prion diseases. There is a surge of evidence that suggests that large protein aggregates of a range of proteins are able to enter cells via macropinocytosis. Our recent work suggests that this process is activated by the binding of aggregates to the neuron cell surface. The current review considers the potential role of cell surface receptors in the triggering of macropinocytosis by protein aggregates and the possibility of utilizing macropinocytosis pathways as a therapeutic target.     

Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea electron microscopical and biochemical analyses of C-phycocyanin and allophycocyanin aggregates

C-phycocyanin and allophycocyanin from the red alga Rhodella violacea were investigated by electron microscopy and biochemical methods using samples taken from the same fractions.The molecular weights of the native biliprotein aggregates C-phycocyanin and allophycocyanin are about 139,000 (140,000) and 130,000 (145,000) as revealed by calibrated gel chromatography, gradient gel electrophoresis and morphological measurements on the basis of an average protein packing density. These molecular weights are direct evidence for a trimeric aggregation form ()₃ of these biliproteins. Independently, their monomers were determined to be about 34,400 (C-phycocyanin) and 33,900 (allophycocyanin).C-phycocyanin and allophycocyanin are ringshaped, six-membered, biliprotein aggregates with dimensions of about 10.2×3.0 nm and 10.0×3.0 nm, respectively. The aggregates are made up of six subunits, 3 and 3, which are assumed to be associated in alternating positions. They are arranged in regular hexagons in C₆ symmetry. Hexameric aggregates ()₆, so far only isolated for C-phycocyanin, originate by face to face association of two trimeric aggregates.