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Abstract
The displacement adsorption enthalpies (ΔH) of the refolding of lysozyme (Lys) denatured by 1.8 mol L–1 guanidine hydrochloride (GuHCl) on a moderately hydrophobic surface at 298 K, pH 7.0 and various (NH4)2SO4 concentrations were determined by using a Micro DSC-III calorimeter. The study shows that the effect of salt concentrations on the three fractions of the enthalpy is that with increasing (NH4)2SO4 concentrations, the molecular conformation enthalpy of the adsorbed Lys has probably no distinct change at 1.8 mol L–1 GuHCl; the adsorption affinity enthalpy (exothermic) becomes more negative; and the dehydration enthalpy (endothermic) decreases. At lower salt concentrations, the dehydration, especially squeezing water molecules led by molecular conformation, which leads to an entropy-driving process, predominates over the adsorption affinity (also including the orderly orientation of molecular conformation), while at higher salt concentrations, the latter is prior to the former for contribution to ΔH and induces an enthalpy-driving process. Also, the optimal NH4)2SO4 concentration favoring refolding and renaturing of Lys denatured by 1.8 mol L–1 GuHCl was found.
Az endoplazmás retikulum (ER) mint metabolikus kompartment funkciói meghatározók az intracelluláris homeosztázis fenntartásában, a belső és külső környezet zavaraihoz történő alkalmazkodásban. A legkülönbözőbb stresszhelyzetek (intracelluláris kalciumegyensúly változása, hypoglycaemia, hypoxia, redox homeosztázis zavara, vírusfertőzés stb.) érintik az ER luminális kompartimentumában zajló fehérjefolding folyamatrendszerét – foldingzavar alakulhat ki, aminek eredményeképpen nem megfelelően tekert, unfolded fehérjék halmozódnak fel. A változatos ER-stresszformák során megjelenő unfolded fehérjék az ER-ből induló különböző jelátviteli utak aktiválását idézik elő, amelyek sajátos, ER-specifikus válaszreakciók sokaságát váltják ki. A különböző stresszorokra adott hasonló jelátviteli válasz, az „unfolded protein response” (UPR) számos teljesen eltérő etiológiájú betegségben (pl. diabetes mellitus, neurodegeneratív betegségek, hepatitis) aktiválódik, így részben közös patomechanizmust jelent. Ezért az ER-stressz megismerése új távlatokat nyithat a terápiában.
Summary
Calorimetric determination of the total enthalpy changes (ΔH i) of guanidine-denatured lysozyme (Lys) during the adsorption with simultaneously refolding on the surface of hydrophobic interaction chromatography packings was carried out at 250.001C. The measured ΔH iin the circumstances should include the changes in the three fractions: adsorption, dehydration and molecular conformation. It was found that when the unfolded Lys molecules are adsorbed and refold on the surface, entropy-driving caused by the dehydration of Lys mainly dominates the foregoing process. The refolding enthalpies of Lys, ΔΔH iwere found to be 10~100 folds higher than that measured in usual solutions.</o:p>
Abstract
Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.
involved. The questions we might wish to address are as follows: what makes a protein fold, and what controls its stability? Once it has folded, what does the protein bind to and what is its function?—questions central to practical applications in
L. pneumophila is an intracellular bacterium that replicates inside a membrane-bound vacuole called Legionella-containing vacuole (LCV), where it plentifully liberates its HtpB chaperonin. From LCV, HtpB reaches the host cell cytoplasm, where it interacts with SAMDC, a cytoplasmic protein required for synthesis of host polyamines that are important for intracellular growth of L. pneumophila. Additionally, cytoplasmic expression of HtpB in S. cerevisiae induces pseudohyphal growth, and in mammalian cells recruits mitochondria to LCV, and modifies actin microfilaments organization. This led us to hypothesize here that HtpB recruits a protein(s) from eukaryotic cells that is involved in the emergence of the aforementioned phenotypes. To identify this protein, a commercially available HeLa cDNA library was screened using a yeast two-hybrid system. Approximately 5×106 yeast clones carrying HeLa cDNA library plasmid were screened. Twenty-one positive clones were identified. DNA sequence analysis revealed that all of these positive clones encoded the mammalian small heat shock protein Hsp10. Based on the fact that chaperonions are required to interact with co-chaperonins to function properly in protein folding, we believe that HtpB recruits the host cell Hsp10 to appropriately interact with SAMDC and to induce the multifunction phenotypes deemed important in L. pneumophila pathogenesis.
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