Under stress: Changes in stress of the polymer matrix in a protein–polymer hybrid material result in changes of conformation of the protein complex, thus resulting in a damage‐reporting material (see picture). The reporter is an engineered chaperonin that covalently entraps a pair of fluorescent proteins. Deformation of the chaperonin leads to a change in fluorophore distance and a change in the fluorescene resonance energy transfer (FRET) signal.
For insight into the solvent structure around protein molecules and its role in phase transformations, we investigate the thermodynamics of crystallization of the rhombohedral form of porcine insulin crystals. We determine the temperature dependence of the solubility at varying concentration of the co-solvent acetone, Cac=0%, 5%, 10%, 15%, and 20%, and find that, as a rule, the solubility of insulin increases as temperature increases. The enthalpy of crystallization, undergoes a stepwise shift from approximately -20 kJ mol(-1) at Cac=0%, 5%, and 10% to approximately -55 kJ mol(-1) at Cac=15% and 20%. The entropy change upon crystallization is approximately 35 J mol(-1) K(-1) for the first three acetone concentrations, and drops to approximately -110 J mol(-1) K(-1) at Cac=15% and 20%. DeltaS degrees cryst>0 indicates release of solvent, mostly water, molecules structured around the hydrophobic patches on the insulin molecules' surface in the solution. As Cac increases to 15% and above, unstructured acetone molecules apparently displace the waters and their contribution to DeltaS degrees cryst is minimal. This shifts DeltaS degrees cryst to a negative value close to the value expected for tying up of one insulin molecule from the solution. The accompanying increase in DeltaH degrees cryst suggests that the water structured around the hydrophobic surface moieties has a minimal enthalpy effect, likely due to the small size of these moieties. These findings provide values of the parameters needed to better control insulin crystallization, elucidate the role of organic additives in the crystallization of proteins, and help us to understand the thermodynamics of the hydrophobicity of protein molecules and other large molecules.
The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFβ1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFβs significantly increased expression of αSMA, SM22α, versican, the TGFβs themselves, and deposition of PGs (P<0.05), with TGFβ1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFβ neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (αSMA), 5HTR2B , TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFβ1 and the TGFβ RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.
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