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PubMed1991

The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli.

The in vitro and in vivo analysis of the ribonuclease E-deficient (rne-) and the altered mRNA stability protein-deficient (ams-) strains of Escherichia coli has demonstrated that they carry mutations in the same structural gene. Strains encoding either thermolabile RNase E (rne-3071) or Ams protein (ams-1) are defective in both rRNA processing and mRNA turnover. Immediately after a shift to the nonpermissive temperature, the chemical decay rate of bulk mRNA is slowed 2- to 3-fold, and within 70 min, precursors to 5S rRNA begin to accumulate. In addition, all of the phenotypes associated with either the rne-3071 or the ams-1 alleles were complemented by a recombinant plasmid carrying ams+. When taken together with previous genetic studies, these results suggest that the role of ribonuclease E in mRNA turnover involves endonucleolytic cleavages at the proposed ACAG(A/U)AUUUG consensus sequence.

Bacterial Proteins↗
PubMed2005

Stability, protein binding and thiol interaction studies on [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt.

Cobalt-alkyne complexes are a new class of potent cytotoxic drugs. The lead compound [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS) showed high effects on several human cancer cell lines. In order to evaluate further the in-vitro properties and reactivity of this substance we performed stability and protein binding studies and investigated the interaction of this complex with 1,2-ethanedithiol, L-cysteine and glutathione. UV-Vis, HPLC and AAS studies showed that the compound was sufficiently stable under in-vitro conditions. Binding to human serum albumin increased from approximately 25% at the beginning to over 50% after 48 h of incubation as determined by ethanol preciptation and size exclusion chromatography. The interaction with thiols resulted in disulfide bond formation of the thiols.

Chromatography, Gel↗
PubMed1988

Genetic studies of protein stability and mechanisms of folding.

In the past few years, analysis of mutationally altered proteins has joined more traditional biophysical methods as an important experimental approach to the study of protein folding. Single amino acid substitutions have been found to reduce the stability of the native state by as much as 3 kcal mol-1, a significant fraction of the marginal stability of the folded conformation, and to significantly alter the rates of folding or unfolding. Mutations can also significantly affect the ability of a protein to fold in vivo. However, at present there is great uncertainty in accounting for or predicting the effects of mutations on stability and folding, even qualitatively. Future studies of mutant proteins are likely to provide further insights into the roles of individual residues and interactions in determining the three-dimensional structures of proteins. For the full potential of this approach to be realized, however, many mutant proteins will have to be systematically studied using structural, thermodynamic, and kinetic methods. The results of these experiments may aid in developing and refining improved theoretical treatments of the energetics of protein conformation.

Drug Stability↗
PubMed1998

Why is p53 protein stabilized in neoplasia? Some answers but many more questions?

The p53 pathway provides a physiological system for integrating signals from diverse insults and eliciting adaptive cellular responses that include (but importantly are not restricted to) growth arrest and apoptosis. Defects in the pathway are prevalent in cancer, most notably being associated with mis-sense mutations in p53 itself. This leads to the inability of p53 to act as a transcription factor and thus to the non-occurrence of downstream events. Recent data indicate that the stability (and hence level) of p53 protein in cells is regulated by its interaction with mdm2: this results in enhanced p53 degradation by ubiquitin-mediated events. Since mdm2 is itself regulated by p53, loss of function of p53 leads to lack of mdm2 and thus to p53 protein accumulation. This provides a mechanistic explanation for the observation that p53 accumulation is associated with neoplasia. It may be that accumulation of p53 in the absence of p53 mutation can occur as a consequence of mdm2 defects, as well as being a physiological response in many situations. Another recent development is the recognition of p53 homologues (p73 alpha, p73 beta, and KET) which have many sequence and probable structural features in common with p53. It seems likely that this will reveal another layer of complexity in the control and regulation of p53 and its role in physiology and pathology.

Humans↗
PubMed2005

Determinants of protein stability and folding: comparative analysis of beta-lactoglobulins and liver basic fatty acid binding protein.

A new energy decomposition approach, aimed at identifying residues playing a folding key role, has been applied here to three homologous proteins, belonging to the calycin superfamily, namely bovine and porcine beta-lactoglobulins and Liver basic fatty acid binding protein, sharing the same beta-barrel fold and different degree of sequence identities. All-atom, explicit solvent molecular dynamics simulations around the native conformation were used to generate, for each of the three proteins, energy maps which were further simplified through eigenvalue decomposition. Analysis of the components of the eigenvector associated with the lowest eigenvalue singled out those residues (hot sites) behaving as strongly interacting and possible nucleation centers. The results fit well with experimental folding data and, especially, with the analysis of side chain-side chain interaction conservation.

Amino Acid Sequence↗
PubMed1999

ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system.

We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

Adenosine Triphosphate↗
PubMed2006

Targeting protein stability with a small molecule.

Both genetic approaches and small-molecule inhibitors can be used to perturb protein function. Banaszynski et al. (2006) now describe a strategy to regulate the stability of a target protein, in which specificity is provided by a genetically encoded tag and temporal control is imparted by a small molecule.

Animals↗
PubMed1992

Histidine-aromatic interactions in barnase. Elevation of histidine pKa and contribution to protein stability.

Aromatic side-chains are found in the vicinity of histidine residues in many proteins and protein complexes. We have studied the interaction between a histidine residue (His18) and aromatic residues at position 94 in barnase. Three different techniques have been applied to show that Trp94 interacts more strongly with the protonated form of His18. The aromatic-histidine interaction stabilizes the protonated form of histidine by 0.8 to 1 kcal mol-1 relative to the unprotonated and, thereby, increases its pKa value. This was shown indirectly from the pH dependence of the stability of the wild-type protein and the mutant Trp94----Leu; and directly from the difference in pKa of His18 between wild-type barnase and the same mutant protein, and from double-mutant cycles that measure the total interaction energy of Trp94 with His18 at both low and high pH. When Trp94 is replaced by other aromatic amino acids, the strength of the interaction decreases in the series His-Trp greater than His-Tyr greater than His-Phe. The interaction is not masked by high salt concentrations. The raising of the pKa value of His18 by interaction with Trp94 is shown to be consistent with solution studies with model compounds. The histidine-aromatic interaction could have implications in binding and catalysis for modulation of the histidine pKa value.

Amino Acid Sequence↗
PubMed2003

An intron is required for dihydrofolate reductase protein stability.

We compared the expression of dihydrofolate reductase minigenes with and without an intron. The levels of protein were significantly higher in the presence of dihydrofolate reductase intron 1. However, mRNA levels in both constructs were comparable. In addition, the RNA transcribed from either construct was correctly polyadenylated and exported to the cytoplasm. The intron-mediated increase in dihydrofolate reductase protein levels was position-independent and was also observed when dihydrofolate reductase intron 1 was replaced by heterologous introns. The translational rate of dihydrofolate reductase protein was increased in transfectants from the intron-containing minigene. In addition, the protein encoded by the intronless construct was unstable and subject to lysosomal degradation, thus showing a shorter half-life than the protein encoded by the intron-containing minigene. We conclude that an intron is required for the translation and stability of dihydrofolate reductase protein.

Alternative Splicing↗
PubMed2000

Protein stabilization through phage display.

RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.

Amino Acid Sequence↗
PubMed1999

Differential protease-mediated turnover of H-NS and StpA revealed by a mutation altering protein stability and stationary-phase survival of Escherichia coli.

The Escherichia coli proteins H-NS is recognized as an important component among the major nucleoid-associated proteins. In studies of E. coli strains with defects in H-NS, we discovered a mutant that phenotypically restored stationary-phase viability (Rsv) of such strains. The Rsv phenotype was the result of a mutation that led to severalfold higher levels of the functionally and structurally related StpA protein. This mutation was a base pair change in the stpA structural gene, and the amino acid substitution in the StpA protein altered its turnover properties, suggesting a role for this residue in a cleavage site for proteolysis. We determined the stability of the StpA and the H-NS proteins and found that the StpA protein was degraded relatively rapidly in strains lacking functional H-NS, whereas H-NS remained stable irrespective of the presence/absence of StpA. Using protease-deficient mutants, we obtained evidence that the Lon protease was responsible for the degradation of StpA. The differential turnover of the nucleoid-associated proteins is suggested to contribute to the regulation of their stoichiometry and ratio in terms of homo- and heteromer formation. We conclude that StpA, in contrast to H-NS, is present mainly in heteromeric form in E. coli.

ATP-Dependent Proteases↗
PubMed2004

The extra fragment of the iron-sulfur protein (residues 96-107) of Rhodobacter sphaeroides cytochrome bc1 complex is required for protein stability.

Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc(1) complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPDelta(96-107), ISP(96-107)A, and ISP(104-107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc(1) complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc(1) activity found in the complement chromatophores. The loss (or decrease) of bc(1) activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c(1) into the membrane, the binding affinity of cytochrome b to cytochrome c(1), or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99.

Amino Acid Sequence↗
PubMed2004

Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability.

Rotavirus VP6 is a highly immunogenic major capsid protein that may be useful as a subunit vaccine. The expression of a bovine group A rotavirus VP6 cDNA was examined in tobacco chloroplasts following particle bombardment. Constructs containing the VP6 cDNA under the control of plastid rrn or psbA promoters, or the Escherichia coli trc promoter, were inserted, together with the aadA selectable marker gene, between the rbcL and accD genes of the tobacco plastid genome. The 40-kDa VP6 protein accumulated to about 3% of total soluble protein in seedlings and young leaves of homoplasmic transplastomic plants containing the VP6 cDNA under the control of the rrn promoter. Lower amounts of VP6 (approximately 0.6% total soluble protein) accumulated in plants containing the VP6 cDNA under the control of the psbA promoter, and VP6 was undetectable in plants containing the VP6 cDNA under the control of the trc promoter. The VP6 protein in chloroplasts was shown to form trimers, as found in the rotavirus virion. However, the amount of VP6 protein declined as the leaves matured, although VP6 transcripts were still present, suggesting that the protein was susceptible to proteolytic degradation in chloroplasts.

Journal Article↗
PubMed2006

Nucleostemin delays cellular senescence and negatively regulates TRF1 protein stability.

Nucleostemin (NS) encodes a nucleolar GTP-binding protein highly enriched in the stem cells and cancer cells. To determine its biological activity in vivo, we generated NS loss- and gain-of-function mouse models. The embryogenesis of homozygous NS-null (NS(-/-)) mice was aborted before the blastula stage. Although the growth and fertility of heterozygous NS-null (NS(+/-)) mice appeared normal, NS(+/-) mouse embryonic fibroblasts (MEFs) had fewer NS proteins, a lower population growth rate, and higher percentages of senescent cells from passage 5 (P5) to P7 than their wild-type littermates. Conversely, transgenic overexpression of NS could rescue the NS(-/-) embryo in a dose-dependent manner, increase the population growth rate, and reduce the senescent percentage of MEFs. Cell cycle analyses revealed increased pre-G1 percentages in the late-passage NS(+/-) MEF cultures compared to the wild-type cultures. We demonstrated that NS could interact with telomeric repeat-binding factor 1 (TRF1) and enhance the degradation but not the ubiquitination of the TRF1 protein, which negatively regulates telomere length and is essential for early embryogenesis. This work demonstrates the roles of NS in establishing early embryogenesis and delaying cellular senescence of MEFs and reveals a mechanism of a NS-regulated degradation of TRF1.

Animals↗
PubMed2003

Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.

Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20 degrees C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJ x mol(-1) in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.

Amides↗
PubMed1985

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.

DNA, Bacterial↗