Stability of Protein Formulations at Subzero Temperatures by Isochoric Cooling.

Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP ß-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.

Obtaining a protocol for extraction of phenolics from açaí fruit pulp through Plackett-Burman design and response surface methodology.

This work aimed to obtain a simplified extraction protocol for simultaneous achievement of total anthocyanin and total phenolic in açaí pulp using a 3-step optimization approach. First, a Plackett-Burman 20 was applied in 16 independent variables selected in literature. Secondly, seven factors pre-selected in the first screening were reassessed using a Plackett-Burman 12. Then, four selected factors; solid/solvent ratio (g:mL), acetone concentration (%), time of extraction in acidified ethanolic solution (min) and ethanol concentration (%) were optimized using a central composite design with response surface methodology. In addition, the optimized protocol were compared with two standardized extraction procedures assessing açaí and grape pulps. The optimized method is effective for the simultaneous extraction of total phenolics and total anthocyanins, allowing representative measurements of free radical-scavenging capacity (DPPH) and trolox equivalent capacity (TEAC) of grape and açaí pulps, with savings of time and reagents, moreover, avoiding the use of methanol.

Sucrose prevents protein fibrillation through compaction of the tertiary structure but hardly affects the secondary structure.

Amyloid fibers, implicated in a wide range of diseases, are formed when proteins misfold and stick together in long rope-like structures. As a natural mechanism, osmolytes can be used to modulate protein aggregation pathways with no interference with other cellular functions. The osmolyte sucrose delays fibrillation of the ribosomal protein S6 leading to softer and less shaped-defined fibrils. The molecular mechanism used by sucrose to delay S6 fibrillation was studied based on the two-state unfolding kinetics of the secondary and tertiary structures. It was concluded that the delay in S6 fibrillation results from stabilization and compaction of the slightly expanded tertiary native structure formed under fibrillation conditions. Interestingly, this compaction extends to almost all S6 tertiary structure but hardly affects its secondary structure. The part of the S6 tertiary structure that suffered more compaction by sucrose is known to be the first part to unfold, indicating that the native S6 has entered the unfolding pathway under fibrillation conditions.

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

Compacting proteins: pros and cons of osmolyte-induced folding.

Biomedical applications of osmolytes, including stabilization of protein-based pharmaceutics, preservation of living biological material and potential therapeutic prescription in vivo, are intimately related to the fact that osmolytes favour the native structure of proteins. The shift towards the native structure is associated to the compaction of the protein by a non-specific mechanism. This compaction is observed mostly for the unfolded state but also for the transition state ensemble and even for the native state. In addition, more stable three-dimensional structures are more stabilized by osmolytes if the overall protein fold is the same indicating that point mutations and osmolytes should share a similar mechanism for protein stabilization. A synergistic effect to increase protein stability between accumulation of osmolytes and protein engineering strategies seems to have operated during evolution. However, the conformational pre-organization of the unfolded state (compaction) induced by osmolytes which increases the folding rate, might lead to the accumulation of off-folding pathway intermediates with non-native structure that delay folding. Also, osmolytes favor protein aggregation as an alternative way to shield protein surfaces from the solvent. The sometimes observed effect of osmolytes on the prevention of protein aggregation is apparent as they only decrease the accumulation of aggregation-competent partially unfolded states.