Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

Multidomain structure of a recombinant streptokinase. A differential scanning calorimetry study

The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.     

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles

Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

人血清白蛋白与镍离子相互作用的热力学研究

1　Introduction　　Serumalbuminproteinsareamongthemosthighlystudiedandappliedinbiochemistry[1～ 4].Albuministhemostabundantproteininbloodplasmaandoneofitsmainfunctionsisbasedonauniqueabilitytobindnumerousendogenousandexogenouscompounds.Duetoitsligandbindingpropertiesalbuminservesasacirculatingdepotofsomemetabolites.Thisdepoteffectisoftenmadeuseofindrugtherapy.　　Humanserumalbumin(HSA)isasinglepeptidechainconsistingof 5 85aminoacids( 6 6 5ku)asdeterminedbyaminoacidsequencestudies[5] andasde…