Protein aggregates as depots for the release of biologically active compounds.

Protein misfolding and aggregation is one of the most serious problems in cell biology, molecular medicine, and biotechnology. Misfolded proteins interact with each other or with other proteins in non-productive or damaging ways. However, a new paradigm arises that protein aggregation may be exploited by nature to perform specific functions in different biological contexts. From this consideration, acceleration of stress-induced protein aggregation triggered by any factor resulting in the formation of soluble aggregates may have paradoxical positive consequences. Here, we suggest that amorphous aggregates can act as a source for the release of biologically active proteins after removal of stress conditions. To address this concept, we investigated the kinetics of thermal aggregation in vitro of yeast alcohol dehydrogenase (ADH) as a model substrate in the presence of two amphiphilic peptides: Arg-Phe or Ala-Phe-Lys. Using dynamic light scattering (DLS) and turbidimetry, we have demonstrated that under mild stress conditions the concentration-dependent acceleration of ADH aggregation by these peptides results in formation of large but soluble complexes of proteins prone to refolding.

Effect of mutations in the beta5-beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11).

The human genome encodes ten different small heat shock proteins, each of which contains the so-called alpha-crystallin domain consisting of 80-100 residues and located in the C-terminal part of the molecule. The alpha-crystallin domain consists of six or seven beta-strands connected by different size loops and combined in two beta-sheets. Mutations in the loop connecting the beta5 and beta7 strands and conservative residues of beta7 in alphaA-, alphaB-crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the beta5-beta7 loop and in the beta7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases.

Effects of arginine on kinetics of protein aggregation studied by dynamic laser light scattering and tubidimetry techniques.

Prevention of undesirable protein aggregation is an extremely important strategy in protein science, medicine, and biotechnology. Arginine is one of the most widely used low molecular weight solution additives effective in suppressing aggregation, assisting refolding of aggregated proteins, and enhancing the solubility of aggregation-prone unfolded molecules in vitro. However, the mechanism of suppression of protein aggregation by arginine is not well understood. To address the mechanism, two model systems have been investigated: protection of alcohol dehydrogenase (ADH) and insulin from heat- and dithiothreitol-induced aggregation, respectively, in the presence of arginine. Using dynamic light scattering (DLS) technique, we have demonstrated the concentration-dependent suppression of light scattering intensity of both ADH and insulin aggregates upon addition of arginine to the incubation medium, a significant effect being revealed in the physiological concentration range of arginine (1-10 mM). DLS studies showed that arginine shifted the populations of nanoparticles with higher hydrodynamic radii to the lower ones, suggesting that the preventive effect of arginine on the protein aggregation process arises because it suppresses intermolecular interactions among aggregation-prone molecules. The results of turbidity measurements were also shown to be consistent with these findings.

Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders.

Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.