Ligand-induced biphasic protein denaturation

The results of a thermodynamic calculation of the excess heat capacity that is based on experimental observations and that incorporates the effects of ligand binding on the two-state, thermal denaturation of a protein are presented. For a protein with a single-binding site on the native species and at subsaturating concentrations of ligand, bimodal or unimodal thermograms were computed merely by assuming a larger or smaller ligand association constant, respectively. The calculated thermograms for this simplified case show the salient features of those observed by differential scanning calorimetry for defatted human albumin monomer in the absence and presence of three ligands for which the protein has higher, intermediate, and lower affinity (Shrake, A., and Ross, P. D. (1988) J. Biol. Chem. 263, 15392-15399). The computation demonstrates that biphasic unfolding can result from a significant increase in the free energy of denaturation (and the transition temperature) during the course of unfolding due to a substantial increase in free ligand concentration caused by the release of bound ligand by denaturing protein. Such ligand-induced biphasic denaturation does not relate to macromolecular substructure but derives from a perturbation, during unfolding, of the ligand binding equilibrium, which is coupled to the equilibrium between the folded and unfolded protein species. Thus, this bimodality is not limited to thermally induced unfolding but is operative independent of the means used to effect denaturation and therefore must be considered when studying any macromolecular folding/unfolding reaction in the presence of ligand.     

Quinotrierixin inhibited ER stress-induced XBP1 mRNA splicing through inhibition of protein synthesis

Quinotrierixin was isolated from microbes as an inhibitor of ER stress-induced XBP1 mRNA splicing, but its mode of action was unclear. We found that quinotrierixin is an inhibitor of protein synthesis, and that the required dose range of quinotrierixin to inhibit ER stress-induced XBP1 mRNA splicing was similar to that to inhibit protein synthesis. Furthermore, we also found that quinotrierixin inhibited the ER stress-induced increases of unfolded protein response-related genes such as GRP78, CHOP, EDEM, ERdj4, and p58(IPK). Thus, we showed that quinotrierixin inhibited the ER stress-induced unfolded protein response, possibly due to its inhibitory activity of protein synthesis.     

Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity

Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene. Parkin protein is characterized by a ubiquitin-like domain at its NH(2)-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppressed unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses.     

Acid denaturation of mustard 12S protein

The effect of low pH on the molecular properties of mustard 12S protein has been studied by the techniques of ultracentrifugation, viscometry, electrophoresis, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism. Ultracentrifugation and electrophoresis experiments indicated dissociation of the protein in the pH range 5.0 to 3.0 and below this pH reaggregation was indicated. Viscosity, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism studies showed that denaturation of the protein occurred between pH 5.0 and 3.0 and refolding at pH values below 3.0.     

Effects of thermal denaturation on protein glycation

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.     

Ammonia aggravates stress-induced gastric mucosal oxidative injury through the cancellation of cytoprotective heat shock protein 70

The relationship between Helicobacter pylori colonization and the formation of stress-induced gastric mucosal injury remains unknown. Since ammonia (NH(3)) is known as one of the injurious factors in H. pylori-colonized gastric mucosa, the present study is designed to investigate the level of stress-induced gastric mucosal oxidative injury with or without intragastric NH(3) overloading. To apply emotional stress, the communication box paradigm was used in the mouse model. Mice (C57BL/6, male) were pretreated with distilled water (responder-H(2)O) or 0.01% NH(3) (responder-NH(3)) through a gastric tube once a day for a week. Emotional stress was then applied to the responder mice for 3 h per day for 3 d by watching and hearing the behavior of the sender mice subjected to electric shocks to the feet (2 mA, 10 s, 50 s interval). After the communication box protocol, the tissue MPO activity, the contents of TBA-reactive substances (TBARS), and the level of gastric mucosal HSP70 were examined. Responder-NH(3) mice developed more severe gastric lesions than the responder-H(2)O subjects. MPO activity and TBARS contents were enhanced significantly in the responder-NH(3) group compared with the responder-H(2)O subjects. Although the contents of HSP70 in the gastric mucosa increased in the responder-H(2)O group compared with the control-H(2)O animals, they were significantly attenuated in the responder-NH(3) mice. Excess intragastric NH(3) was able to enhance the formation of emotional stress-induced gastric mucosal lesions. This injury may be associated with the enhanced production of oxygen free radicals from accumulated neutrophils under the NH(3)-mediated cancellation of gastric mucosal cytoprotective HSP70.     

Bone morphogenetic protein

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.     

Carnosine promotes the heat denaturation of glycated protein

Glycation alters protein structure and decreases biological activity. Glycated proteins, which accumulate in affected tissue, are reliable markers of disease. Carnosine, which prevents glycation, may also play a role in the disposal of glycated protein. Carnosinylation tags glycated proteins for cell removal. Since thermostability determines cell turnover of proteins, the present study examined carnosine's effect on thermal denaturation of glycated protein using cytosolic aspartate aminotransferase (cAAT). Glycated cAAT (500 microM glyceraldehyde for 72h at 37 degrees C) increased the T(0.5) (temperature at which 50% denaturation occurs) and the Gibbs free energy barrier (DeltaG) for denaturation. The enthalpy of denaturation (DeltaH) for glycated cAAT was also higher than that for unmodified cAAT, suggesting that glycation changes the water accessible surface. Carnosine enhanced the thermal unfolding of glycated cAAT as evidenced by a decreased T(0.5) and a lowered Gibbs free energy barrier. Additionally, carnosine decreased the enthalpy of denaturation, suggesting that carnosine may promote hydration during heat denaturation of glycated protein.     

Snap denaturation reveals dimerization by AraC-like protein Rns

Here we show that the Rns regulator of Escherichia coli dimerises in vivo and in vitro. Furthermore, we demonstrate that Rns forms aggregates in vitro and describe a methodology to ameliorate aggregation thus permitting the analysis of Rns by cross-linking.     

alpha-B- and alpha-A-crystallin prevent irreversible acidification-induced protein denaturation

alpha-Crystallin (alpha), a major structural protein of the mammalian lens, is a large, physically heterogeneous macromolecule with an average molecular weight of approximately 800 kDa and is composed of two 20-kDa polypeptides designated as alphaA and alphaB. A line of evidence strongly suggests that alphaB may have an essential nonlenticular function. Here it is demonstrated that alphaB can bind partially denatured enzymes effectively at acidic pH and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, when the inactive luciferase bound to alphaB was treated with reticulocyte lysate (a rich source of molecular chaperones) and an ATP-generating system, more than 50% of the original luciferase activity could be recovered. Somewhat less activation was observed when alphaA-bound enzyme or the alpha-bound enzyme was renatured similarly. The overall results suggest that alpha acts as a chaperone to stabilize denaturing proteins at acidic pH so that at a later time they can be reactivated by other chaperones.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Interpretation of thermograms of protein denaturation in non-equilibrium conditions

Data are presented concerning the effect of heating rate on the denaturation parameters of small and oligomeric globular proteins: Kunitz trypsin inhibitor from soybeans and 1,5-Ribulose Bisphosphate Carboxylase from tobacco leaves. Substantional dependence of denaturation temperature on the heating rate reflects non-equilibrium pattern of denaturation of these proteins under experimental conditions. To interpret these data a kinetic approach is proposed, which permits determination of equilibrium value of the denaturation temperature and of the constant of de- and renaturation rate. The conformation transitions in the proteins studied are shown to be relatively slow processes. Their rate is comparable to the velocity of temperature change in a calorimeter, which is the cause of non-equilibrium effects in a calorimetric experiment.     

Backbone and side-chain contributions in protein denaturation by urea

Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.