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Results for “Protein Stability”

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PubMed1997

Photoassembly of the photosystem II (Mn)4 cluster in site-directed mutants impaired in the binding of the manganese-stabilizing protein.

Photoactivation is the light-dependent ligation of Mn2+ into the H2O oxidation complex of photosystem II (PSII) and culminates in the formation of an enzymatically active complex containing Ca2+ and four Mn>/=3+. Previous kinetic analysis demonstrated that the genetic removal of the extrinsic manganese-stabilizing protein (MSP) increases the quantum yield of photoactivation 4-fold relative to that of the wild type, consistent with the hypothesis that MSP hinders access of Mn2+ to a site of photoligation [Burnap, R. L., et al. (1996) Biochemistry35, 874-882]. In this report, several Synechocystis sp. PCC6803 mutants with defined amino acid substitutions in the N-terminal region of MSP or the e-loop of intrinsic PSII protein CP47 [Putnam-Evans, C., et al. (1996) Biochemistry 35, 4046-4053] were characterized in terms of the binding of MSP to the intrinsic portion of the PSII complex and in terms of photoactivation kinetics. The charge-pair switch mutation, Arg384Arg385 --> Glu384Glu385 in the lumenal e-loop of CP47 (CP47 RR384385EE), exhibited the most severe impairment of MSP binding, whereas the Arg384Arg385 --> Gly384Gly385 (CP47 RR384385GG) mutation caused a more moderate impairment in binding. Single-substitution mutations at the highly conserved Asp9 or Asp10 positions in the amino-terminal region of MSP also resulted in a reduced binding affinity, but not as severe as that in CP47 RR384385EE. The relative quantum yield of photoactivation of hydroxylamine-extracted mutant PSII was generally found to correlate with the degree of MSP binding impairment, with the CP47 RR384385 mutants exhibiting the highest quantum yields. A two-locus, double-mutant construct involving deletion of MSP in the CP47 RR384385EE background was found to be only slightly more impaired in H2O oxidation activity than either of the corresponding single-locus mutant derivatives, indicating that mutations at these genetically separate loci encode physically interacting products affecting the same reaction parameter during H2O oxidation. Taken together, the results reinforce the concept that MSP interacts with the e-loop of CP47 at Arg384Arg385 and that disruption of this interaction causes significant alterations of the site of H2O oxidation in terms of assembly and enzymatic activity of the Mn cluster.

Darkness↗

PubMed2001

Quantitative protein stability measurement in vivo.

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.

Escherichia coli↗

PubMed1993

A conserved 8 bp motif (GCYATCAY) in the 3'UTR of transition protein 2 as a putative target for a transcript stabilizing protein factor.

The 3'UTR of the transition protein 2 gene of several mammalian species was sequenced and the transcript level of this gene was determined by Northern blots. In all species tested so far, a Northern blot detectable transcript level was associated with the presence of a conserved 8 bp motif (GCYATCAY) 50 bp downstream of the stop codon. RNA-bandshift experiments indicate that this region is capable of binding a cytosolic protein factor from rat testis. These findings support our hypothesis that the low transcript level of the transition protein 2 gene in human is due to insufficient storage of the mRNA as ribonucleic/protein particle.

Animals↗

PubMed2005

A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro.

Nocturnal melatonin production is reportedly controlled by the rhythms of serotonin N-acetyltransferase (NAT, or arylalkylamine N-acetyltransferase). While analyzing the melatonin synthetic pathways of Long Evans cinnamon (LEC) rats mutant for PINA, a pineal night-specific ATPase defective in Wilson disease, we discovered that NAT activity and protein levels are greatly reduced in LEC rats, and that the highly conserved histidine 28 is mutated to tyrosine. To study the effect of H28Y, we isolated a new strain of rat termed LPN that is mutant for NAT but wildtype for both PINA and coat color. Compared with control rats, the LPN rats displayed low NAT protein levels and enzyme activities. These results suggest that the H28Y mutation in NAT is the cause of reduced NAT levels in vivo. The identical H28Y mutation was also found in Sprague-Dawley rats from Zivic-Miller, suggesting it may be a common mutation in rodents. When analyzed in bacterial cells and HEK293 cells, the mutation resulted in reduction of both NAT protein stability and catalytic activity, confirming that the in vivo NAT phenotype in LPN rats was due to the H28Y mutation. Further analysis of the NAT-H28Y will focus on the mechanisms of the increased degradation both in vitro and in vivo, which will facilitate our understanding of how melatonin synthesis is controlled at the molecular level.

Adenosine Triphosphatases↗

PubMed2004

Complex coacervation of lysozyme and heparin: complex characterization and protein stability.

PURPOSE: To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability. METHODS: Insoluble lysozyme-heparin complexes were formed at pH 7.2 and the binding stoichiometry determined using a solution depletion method. Protein structure was determined by infrared spectroscopy and intrinsic fluorescence. Protein stability was evaluated using differential scanning calorimetry and followed in a 12-weeks storage stability study at 37 degrees C. RESULTS: Binding stoichiometry between heparin and lysozyme was found to be dependent on ionic strength of the solution. At low ionic strength (I approximately 0.01) about 11 lysozyme molecules could bind to a 17 kDa heparin chain, 3 to a 6 kDa chain, and less than 2 to a 3 kDa chain. At higher ionic strength (I approximately 0.1), only 7 lysozyme molecules could bind to a 18 kDa heparin chain.. Above ionic strengths of approximately 0.32 M, no insoluble complexes were observed. Infrared spectroscopy and intrinsic fluorescence did not show any major changes in protein structure upon complexation to heparin. In contrast, differential scanning calorimetry showed a large decrease in the melting temperature of the protein, from 77 degrees C to 61 degrees C. Moreover, after 12-weeks storage at 37 degrees C, only 60% protein recovery was observed for the complexes, with no loss of protein for the uncomplexed protein. CONCLUSIONS: Heparin has multiple binding sites for lysozyme, amounting to at most one lysozyme molecule per 3 disaccharide units of heparin. Complexation decreased lysozyme stability, suggesting that heparin has a higher affinity for the unfolded state than the native state. Similar destabilization may occur for other proteins upon interaction with highly charged polymeric compounds or surfaces.

Animals↗

PubMed1998

p21(waf1) mRNA contains a conserved element in its 3'-untranslated region that is bound by the Elav-like mRNA-stabilizing proteins.

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein we have localized a high affinity HuD binding site to a 42-nucleotide region within a U-rich tract in the 3'-untranslated region p21(waf1) mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21(waf1) mRNA. p21(waf1) is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3'-untranslated region of p21(waf1) provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle.

Animals↗

PubMed1996

Electrostatic interaction between two domains of isocitrate dehydrogenase from Thermus thermophilus is important for the catalytic function and protein stability.

The role of electrostatic interaction between Lys96 and Glu147 of isocitrate dehydrogenase from Thermus thermophilus was investigated by site-directed mutagenesis. These two residues are located near the active site and involved in the interdomain interaction. Analyses of the catalytic properties and thermostability of the Glu147Gln mutant revealed that this interaction plays important roles in catalytic function and protein stability.

Amino Acid Sequence↗

PubMed1998

Dependence of protein stability on the structure of the denatured state: free energy calculations of I56V mutation in human lysozyme.

Free energy calculations were carried out to understand the effect of the I56V mutation of human lysozyme on its thermal stability. In the simulation of the denatured state, a short peptide including the mutation site in the middle is employed. To study the dependence of the stability on the denatured-state structure, five different initial conformations, native-like, extended, and three random-coil-like conformations, were examined. We found that the calculated free energy difference, DeltaDeltaGcal, depends significantly on the structure of the denatured state. When native-like structure is employed, DeltaDeltaGcal is in good agreement with the experimental free energy difference, DeltaDeltaGexp, whereas in the other four models, DeltaDeltaGcal differs sharply from DeltaDeltaGexp. It is therefore strongly suggested that the structure around the mutation site takes a native-like conformation rather than an extended or random-coil conformation. From the free energy component analysis, it has been shown that free energy components originating from Lennard-Jones and covalent interactions dominantly determine DeltaDeltaGcal. The contribution of protein-protein interactions to the nonbonded component of DeltaDeltaGcal is about the same as that from protein-water interactions. The residues that are located in a hydrophobic core (F3, L8, Y38, N39, T40, and I89) contribute significantly to the nonbonded free energy component of DeltaDeltaGcal. We also propose a general computational strategy for the study of protein stability that is equally conscious of the denatured and native states.

Enzyme Stability↗

PubMed2000

Protein stability in extremophilic archaea.

Extremophilic microorganisms have adapted their molecular machinery to grow and thrive under the most adverse environmental conditions. These microorganisms have found their natural habitat at the boiling and freezing point of water, in high salt concentration and at extreme pH values. The extremophilic proteins, selected by Nature to withstand this evolutionary pressure, represent a wide research field for scientists from different disciplines and the study of the determinants of their stability has been an important task for basic and applied research. A surprising conclusion emerges from these studies: there are no general rules to achieve protein stabilization. Each extremophilic protein adopts various strategies and the outstanding adaptation to extreme temperature and solvent conditions is realized through the same weak electrostatic and hydrophobic interactions among the ordinary amino acid residues which are also responsible for the proper balance between protein stability and flexibility in mesophilic proteins.

Adaptation, Physiological↗

PubMed1994

Protein stabilization by hydrophobic interactions at the surface.

The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis.

Bacterial Proteins↗

PubMed2001

Legume lectin family, the 'natural mutants of the quaternary state', provide insights into the relationship between protein stability and oligomerization.

Legume lectins family of proteins, despite having the same 'jelly roll' tertiary structural fold at monomeric level, exhibit considerable variation in their quaternary structure arising out of small changes in their sequence. Nevertheless, their folding behavior and stability correlates very well with their patterns of assembly into dimers and tetramers. A conservation of their fold during evolution, its wide distribution in many protein families together with the availability of structural information on them make them interesting as proteins to explore the effect of inter- versus intra-subunit interactions in the stability of multimeric proteins. Additionally, as 'natural mutants' of quaternary association, proteins of legume lectin family provide interesting paradigms for studies addressing the effect of subunit oligomerization on the stability, folding and function as well as the evolution of multimeric structures.

Amino Acid Sequence↗

PubMed1998

Erythropoietin loaded microspheres prepared from biodegradable LPLG-PEO-LPLG triblock copolymers: protein stabilization and in-vitro release properties.

Biodegradable microspheres containing recombinant human Erythropoietin (EPO) were prepared from ABA triblock copolymers, consisting of hydrophobic poly(l-lactic-co-glycolic acid) A blocks and hydrophilic polyethylenoxide (PEO) B blocks. Different polymer compositions were studied for the microencapsulation of EPO using a modified double-emulsion process (W/O/W). The encapsulation efficiency for EPO, ranging from 72% to 99% was quite acceptable. The formation of high molecular weight EPO aggregates, however, was higher than in poly(d,l-lactide-co-glycolide) (PLG) microparticles. Using different excipients with known protein stabilizing properties, such as Bovine Serum Albumin (BSA), Poly-l-Histidine (PH), Poly-l-Arginine (PA) or a combination of PA with Dextran 40 (D40), the EPO aggregate content was significantly reduced to <5% of the encapsulated EPO. In contrast to PLG, ABA triblockcopolymers containing >7 mol % PEO, allowed a continuous release of EPO from microspheres for up to 2 weeks under in-vitro conditions. The release profile was comparable to FITC-Dextran 40 kDa (FD 40) loaded microspheres in the initial release phase, while EPO release was leveling off at later time points. BSA additionally prolonged the EPO release, while blends of PLG and PEO did not generate continuous EPO release profiles. LPLG-PEO-LPLG triblock-copolymers (35 mol % PEO; 30 kDa) in combination with 5% BSA yielded both an acceptable level of EPO aggregates and a continuous release profile under in-vitro conditions for up to 2 weeks. The formation of EPO aggregates at later time points is probably induced by acidic cleavage products of the biodegradable polymer and requires further optimization of the ABA polymer composition.

Biocompatible Materials↗

PubMed2003

Anoxic induction of ATF-4 through HIF-1-independent pathways of protein stabilization in human cancer cells.

Hypoxia is a key factor in tumor development, contributing to angiogenesis and radiotherapy resistance. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor regulating the response of cancer cells to hypoxia. However, tumors also contain areas of more severe oxygen depletion, or anoxia. Mechanisms for survival under anoxia are HIF-1alpha independent in Caenorhabditis elegans and, thus, differ from the hypoxic response. Here we report a differential response of cancer cells to hypoxia and anoxia by demonstrating the induction of activating transcription factor-4 (ATF-4) and growth arrest DNA damage 153 (GADD153) protein specifically in anoxia and the lack of induction in hypoxia. By applying RNAi, ATF-4 induction in anoxia was shown to be independent of HIF-1alpha, and desferrioxamine mesylate (DFO) and cobalt chloride induced HIF-1alpha but not ATF-4 or GADD153. Furthermore, the inductive response of ATF-4 and GADD153 was not related to alterations in or arrest of mitochondrial respiration and was independent of von Hippel-Lindau (VHL) disease mutations. In reoxygenated anoxic cells, ATF-4 had a half-life of less than 5 minutes; adding the proteasome inhibitor to normoxic cells up-regulated ATF-4 protein. Extracts from primary human tumors demonstrated more ATF-4 expression in tumors near necrotic areas. Thus, this study demonstrates a novel HIF-1alpha-independent anoxic mechanism that regulates ATF-4 induction at the protein stability level in tumor cells.

Activating Transcription Factor 4↗

PubMedDate not supplied

Pressure shift freezing of pork muscle: effect on color, drip loss, texture, and protein stability.

Cylindrical specimens (50 mm diameter and 160 mm length) of fresh pork muscle (boneless rib portions) packed in plastic bags were frozen by pressure shift freezing (PSF) at 100, 150, and 200 MPa, air blast freezing (ABF), and liquid immersion freezing (LIF). Temperature and phase transformations of the muscle tissue were monitored during the freezing process at three locations: center, midway between the center and the surface, and near the surface. Pork muscle quality changes [color, drip loss (both thawing and cooking), texture (shear force), and protein stability (DSC thermal profiles)] were evaluated after thawing the frozen samples at room temperature (20 degrees C). Employing pressures above 150 MPa caused very significant (P < 0.01) color changes in pork muscle during the PSF process. The PSF process reduced thawing drip loss of pork muscle but did not cause obvious changes in total drip loss following thawing and subsequent cooking. PSF at 150 and 200 MPa resulted in considerable denaturation of myofibrillar proteins of pork muscle. The PSF process also caused an increase in the pork muscle toughness as compared with that of unfrozen, ABF, and LIF samples.

Animals↗

PubMed2002

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate.

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).

Amino Acid Sequence↗

PubMed1996

Amino-acid substitutions in a surface turn modulate protein stability.

A surface turn position in a four-helix bundle protein, Rop, was selected to investigate the role of turns in protein structure and stability. Although all twenty amino acids can be substituted at this position to generate a correctly folded protein, they produce an unusually large range of thermodynamic stabilities. Moreover, the majority of substitutions give rise to proteins with enhanced thermal stability compared to that of the wild type. By introducing the same twenty mutations at this position, but in a simplified context, we were able to deconvolute intrinsic preferences from local environmental effects. The intrinsic preferences can be explained on the basis of preferred backbone dihedral angles, but local environmental context can significantly modify these effects.

Amino Acids↗

PubMed2000

Structural studies on some dityrosine-cross-linked globular proteins: stability is weakened, but activity is not abolished.

We have carried out conformational and stability studies on three proteins that have previously been shown to undergo dityrosine (DT) cross-linking. They include the monomers and dimers of DT-cross-linked calmodulin and the dimers of bovine pancreatic ribonuclease A and bovine eye lens gammaB-Crystallin. In each of these cases, we find the secondary and tertiary structure of the parent protein to be largely maintained. The DT dimer is, however, weaker than the parent. In this sense, the properties of these DT dimers are somewhat similar to those of glutaraldehyde-cross-linked protein crystals. In contrast, the intramolecularly DT-linked monomeric protein that we studied (DT monomer of calmodulin) is seen to have suffered greater changes in its conformation and stability. These results gain significance in light of the growing identification of DT formation as a marker of oxidative stress, aging, and disease.

Calmodulin↗

PubMed1998

Glutamine synthetase expression in rat lung is regulated by protein stability.

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500-700%, GS activity levels rise only 20-45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% (P < 0.01) and decreased lung tissue Gln levels by 70% (P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold (P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% (P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold (P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction (P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.

Animals↗