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PubMed2001

Probing protein stabilization by glycerol using electrospray mass spectrometry.

This study shows that electrospray ionization mass spectrometry (ESI-MS), combined with a heated turbo ion-spray interface, allows monitoring protein stabilization by glycerol in solution. Measurements obtained with the two proteins lysozyme and cytochrome c are presented. The observed mass-to-charge (m/z) distributions reveal the stabilizing effect of the additive on the protein conformations against temperature and acid-induced unfolding, as well as against denaturation by acetonitrile. The data obtained with lysozyme allow detection of minor conformational changes upon glycerol addition to the native protein, and suggest that the protein structure in the presence of the additive is slightly compressed compared with its state in water. This result corroborates previous evidence obtained by nuclear magnetic resonance. It is also shown that analysis of the m/z distributions obtained by ESI-MS can lead to detection of partially folded and partially populated states in protein samples.

Animals↗
PubMed2005

Neural network-based prediction of mutation-induced protein stability changes in Staphylococcal nuclease at 20 residue positions.

Protein-based therapeutics are playing an increasingly important role in the treatment of diseases, including diabetes and cancer. The viability of these treatments, however, are highly dependent on the stability of the therapeutic, since stability affects both the shelf life of the therapeutic as well as its active life in the body. Stability engineering can, therefore, be used to increase the effectiveness of protein-based therapeutics. Computational methods of protein stability prediction have been under development for about a decade, but complex molecular interactions make stability prediction difficult and computationally intensive. A rapid computational method of protein stability prediction is developed using feed-forward neural networks and used to predict mutation-induced stability changes in Staphylococcal nuclease. The input to the neural network consisted of sequences of evolutionarily based amino acid similarity scores that were obtained through the comparison of the amino acids in a mutation containing sequence to their positional counterparts in the baseline wild-type amino acid sequence. A training set was created which consisted of similarity score sequences, for which the stabilities of the corresponding amino acid sequences were known, paired with the relative stabilities of the sequences to that of the baseline. Back-propagation of error was used to train the network to output accurate relative stability scores for the sequences in the training set. Neural network-based relative stability predictions for 55 sequences containing mutation combinations not found in the training set had an accuracy of 92.8%.

Amino Acid Sequence↗
PubMed2003

AMP-activated protein kinase regulates HNF4alpha transcriptional activity by inhibiting dimer formation and decreasing protein stability.

AMP-activated protein kinase (AMPK) is the central component of a cellular signaling system that regulates multiple metabolic enzymes and pathways in response to reduced intracellular energy levels. The transcription factor hepatic nuclear factor 4alpha (HNF4alpha) is an orphan nuclear receptor that regulates the expression of genes involved in energy metabolism in the liver, intestine, and endocrine pancreas. Inheritance of a single null allele of HNF4alpha causes diabetes in humans. Here we demonstrate that AMPK directly phosphorylates HNF4alpha and represses its transcriptional activity. AMPK-mediated phosphorylation of HNF4alpha on serine 304 had a 2-fold effect, reducing the ability of the transcription factor to form homodimers and bind DNA and increasing its degradation rate in vivo. These results demonstrate that HNF4alpha is a downstream target of AMPK and raise the possibility that one of the effects of AMPK activation is reduced expression of HNF4alpha target genes.

AMP-Activated Protein Kinases↗
PubMed1995

Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins.

Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin beta subunit results in the complete absence of the alpha subunit and the linkers. Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain. Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit. Phycocyanin assembly begins with the formation of the alpha beta heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods. The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo.

Amino Acid Sequence↗
PubMed1991

Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles.

The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 kcal/mol to protein stability. There is such an aromatic pair on the solvent-exposed face of the first alpha-helix of barnase, the small ribonuclease from Bacillus amyloliquefaciens. The edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13. The two residues have been mutated both singly and pairwise to alanine, and their free energies of unfolding determined by denaturation with urea. Application of the double-mutant cycle analysis gives an interaction energy of -1.3 kcal/mol for the aromatic pair in the folded protein relative to solvation by water in the unfolded protein. This value is similar to that calculated from the change in surface-accessible area between the rings on the formation of the pair. Analysis of a further double-mutant cycle in which the Tyr residues are mutated to Phe indicates that the aromatic-aromatic interactions of Tyr/Tyr and Phe/Phe make identical contributions to protein stability. However, Tyr is preferred to Phe by 0.3(+/- 0.04) kcal/mol at the solvent-exposed face of the alpha-helix.

Amino Acid Sequence↗
PubMed2002

Plk phosphorylation regulates the microtubule-stabilizing protein TCTP.

The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

Amino Acid Motifs↗
PubMed2005

Regulation of the p73 protein stability and degradation.

p73, a homologue to the tumor suppressor gene p53, is involved in tumorigenesis, though its specific role remains unclear. The gene has two distinct promoters which allow the formation of two protein isoforms with opposite effects: full-length transactivating (TA) p73 shows pro-apoptotic effects, while the shorter DeltaNp73, which lacks the N-terminal transactivating domain, has an evident anti-apoptotic function. Unlike p53, the p73 gene is rarely mutated in human cancers. However, alterations in the relative levels of TA and DeltaNp73 have been shown to correlate with prognosis in several human cancers, suggesting that the fine regulation of these two isoforms is of pivotal importance in controlling proliferation and cell death. Much effort is currently focused on the elucidation of the mechanisms that differentially control TA and DeltaNp73 activity and protein stability, a process complicated by the finding that both proteins are regulated by a similar suite of complex post-translational modifications that include ubiquitination, sequential phosphorylation, prolyl-isomerization, recruitment into the PML-nuclear body (PML-NB), and acetylation. Here we shall consider the main regulatory partners of p73, with particular attention to the recently discovered Itch- and Nedd8-mediated degradation pathways, along with the emerging roles of PML, p38 MAP kinase, Pin1, and p300 in p73 transcriptional activation, and possible mechanisms for the differential regulation of the TAp73 and DeltaNp73 isoforms.

Apoptosis↗
PubMed1997

Predicting protein stability changes upon mutation using database-derived potentials: solvent accessibility determines the importance of local versus non-local interactions along the sequence.

For 238 mutations of residues totally or partially buried in the protein core, we estimate the folding free energy changes upon mutation using database-derived potentials and correlate them with the experimentally measured ones. Several potentials are tested, representing different kinds of interactions. Local interactions along the chain are described by torsion potentials, based on propensities of amino acids to be associated with backbone torsion angle domains. Non-local interactions along the sequence are represented by distance potentials, derived from propensities of amino acid pairs or triplets to be at a given spatial distance. We find that for the set of totally buried residues, the best performing potential is a combination of a distance potential and a torsion potential weighted by a factor of 0.4; it yields a correlation coefficient between computed and measured changes in folding free energy of 0.80. For mutations of partially buried residues, the best potential is a combination of a torsion potential and a distance potential weighted by a factor of 0.7, and for the previously analysed mutations of solvent accessible residues, it is a torsion potential taken individually; the respective correlation coefficients reach 0.82 and 0.87. These results show that distance potentials, dominated by hydrophobic interactions, represent best the main interactions stabilizing the protein core, whereas torsion potentials, describing local interactions along the chain, represent best the interactions at the protein surface. The prediction accuracy reached by the distance potentials is, however, lower than that of the torsion potentials. A possible reason for this is that distance potentials would not describe correctly the effect on protein stability due to cavity formation upon mutating a large into a small amino acid. Last but not least, our results indicate that although local interactions, responsible for secondary structure formation, do not dominate in the protein core, they are not negligible for all that. They have a significant weight in the delicate balance between all the interactions that ensure protein stability.

Animals↗
PubMed2002

N-terminal truncations of manganese stabilizing protein identify two amino acid sequences required for binding of the eukaryotic protein to photosystem II and reveal the absence of one binding-related sequence in cyanobacteria.

Manganese stabilizing protein (MSP) is an intrinsically disordered extrinsic subunit of photosystem II that regulates the stability and kinetic performance of the tetranuclear manganese cluster that oxidizes water to oxygen. An earlier study showed that deletion of the (1)E-(3)G domain of MSP caused no loss of activity reconstitution, whereas deletion of the (4)K-(10)E domain reduced binding of the protein from 2 to 1 mol of MSP/mol of photosystem II and lowered activity reconstitution to about 50% of the control value [Popelkova et al. (2002) Biochemistry 41, 2702-2711]. In this work we present evidence that deletion of 13 or 14 amino acid residues from the MSP N-terminus (mutants DeltaS13M and DeltaK14M) does not interfere either with functional binding of one copy of MSP to photosystem II or with reconstitution of oxygen evolution activity to 50% of the control level. Both of these mutants exhibit nonspecific binding to photosystem II at higher protein concentrations. Truncation of the MSP sequence by 18 amino acids (mutant DeltaE18M), however, causes a loss of protein binding and activity reconstitution. This result demonstrates that the N-terminal domain (15)T-(18)E is required for binding of at least one copy of MSP to photosystem II. Analyses of CD spectra reveal changes in the structure of DeltaE18M (loss of beta-sheet, gain of unordered structure). Use of the information gained from these experiments in analyses of N-terminal sequences of MSP from a number of species indicates that higher plants and algae possess two recognition domains that are required for MSP binding to PSII, whereas cyanobacteria lack the first N-terminal domain found in eukaryotes. This may explain the absence of a second copy of MSP in the crystal structure of PSII from Synechococcus elongatus [Zouni et al. (2001) Nature 409, 739-743].

Amino Acid Sequence↗
PubMed2002

Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems.

Previous studies have established that the selective precipitation of a less soluble buffer component during freezing can induce a significant pH shift in the freeze concentrate. During freezing of sodium phosphate solutions, crystallization of the disodium salt can produce a pH decrease as great as 3 pH units which can dramatically affect protein stability. The objective of our study was to determine how the presence of glycine (0-500 mM), a commonly used bulking agent in pharmaceutical protein formulations, affects the pH changes normally observed during freezing in sodium phosphate buffer solutions and to determine whether these pH changes contribute to instability of model proteins in glycine/phosphate formulations. During freezing in sodium phosphate buffers, the presence of glycine significantly influenced the pH. Glycine at the lower concentrations (< or = 50 mM) suppressed the pH decrease normally observed during freezing in 10 and 100 mM sodium phosphate buffer, possibly by reducing the nucleation rate of salt and thereby decreasing the extent of buffer salt crystallization. The presence of glycine at higher concentration (> 100 mM) in the sodium phosphate buffer resulted in a more complete crystallization of the disodium salt as indicated by the frozen pH values closer to the equilibrium value (pH 3.6). Although high concentrations of glycine can facilitate more buffer salt crystallization and these pH shifts may prove to be potentially damaging to the protein, glycine, in its amorphous state, can also act to stabilize a protein via the preferential exclusion mechanism.

Ammonium Sulfate↗
PubMed2002

Assessment of protein release kinetics, stability and protein polymer interaction of lysozyme encapsulated poly(D,L-lactide-co-glycolide) microspheres.

Using lysozyme as a model protein, this study investigated protein stability, protein--polymer interaction in different release media and their influence on protein release profile and in vitro--in vivo correlation. Lysozyme was microencapsulated into PLGA 50:50 by a double emulsion--solvent extraction/evaporation method. Protein stability, protein--PLGA adsorption and protein in vitro release were studied in various test media. Differential scanning calorimetry analysis showed lysozyme to be most conformationally stable in pH 4.0 acetate buffer with highest T(m) at 77.2 degree C and DeltaH(cal) 83.1 kcal/mol. Lysozyme exhibited good stability in pH 2.5 glycine buffer with T(m) at 63.8 degree C and DeltaH(cal) 69.9 kcal/mol. In pH 7.4 phosphate-buffered saline (PBS), lysozyme showed a trend toward aggregation when the temperature was elevated. When PLGA polymer was incubated with lysozyme in the various buffers, adsorption was found to occur in PBS only. The adsorption severely limited the amount of lysozyme available for release from microspheres, resulting in slow and incomplete release in PBS. In contrast, the release of the microspheres in acetate and glycine buffers was complete within 40 and 70 days, respectively. Radiolabeled lysozyme blood levels in rats from the microspheres correlated qualitatively well with in vitro release in glycine buffer as a release medium. This study suggests that protein stability and adsorption are critical factors controlling protein release kinetics and in vitro--in vivo correlation of PLGA microspheres.

Adsorption↗
PubMed2002

Protein stability: the value of 'old literature'.

The concepts of protein structure and function have been subjects of intensive study throughout the 20th century; they continue to fascinate present-day scientists. Our understanding received a major boost when it was realised during the 1960s, that the physical properties of water play a major role in determining the stability of native proteins in vitro. This recognition changed the emphasis of physicochemical studies towards 'hydration', i.e. protein-water interactions. A rigorous quantitative description of 'hydration' still escapes us, but several semi-quantitative treatments, some with predictive potential, are now available and can account for the marginal stabilities of native proteins in aqueous solvent environments. This article charts the progress achieved during the latter half of the 20th century, which in present day parlance is termed 'old literature'. The thesis is advanced that the common practice of uncritically equating 'recent literature' with 'progress' is of dubious value. In the general area of in vitro protein stability some recent developments seem questionable and have yet to stand the test of time before their usefulness or validity can be accepted.

Animals↗
PubMed1999

Role of structural and sequence information in the prediction of protein stability changes: comparison between buried and partially buried mutations.

Predicting mutation-induced changes in protein stability is one of the greatest challenges in molecular biology. In this work, we analyzed the correlation between stability changes caused by buried and partially buried mutations and changes in 48 physicochemical, energetic and conformational properties. We found that properties reflecting hydrophobicity strongly correlated with stability of buried mutations, and there was a direct relation between the property values and the number of carbon atoms. Classification of mutations based on their location within helix, strand, turn or coil segments improved the correlation of mutations with stability. Buried mutations within beta-strand segments correlated better than did those in alpha-helical segments, suggesting stronger hydrophobicity of the beta-strands. The stability changes caused by partially buried mutations in ordered structures (helix, strand and turn) correlated most strongly and were mainly governed by hydrophobicity. Due to the disordered nature of coils, the mechanism underlying their stability differed from that of the other secondary structures: the stability changes due to mutations within the coil were mainly influenced by the effects of entropy. Further classification of mutations within coils, based on their hydrogen-bond forming capability, led to much stronger correlations. Hydrophobicity was the major factor in determining the stability of buried mutations, whereas hydrogen bonds, other polar interactions and hydrophobic interactions were all important determinants of the stability of partially buried mutations. Information about local sequence and structural effects were more important for the prediction of stability changes caused by partially buried mutations than for buried mutations; they strengthened correlations by an average of 27% among all data sets.

Amino Acids↗
PubMed2004

Comparison between catalase-peroxidase and cytochrome c peroxidase. The role of the hydrogen-bond networks for protein stability and catalysis.

A detailed resonance Raman and electronic absorption investigation has been carried out on a series of novel distal and proximal variants of recombinant catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. In particular, variants of the distal triad Pro-Asp-Asn and the proximal triad His-Asp-Trp have been studied in their ferric and ferrous states at various pH. The data suggest marked differences in the structural role of the conserved residues and hydrogen-bond networks in KatG and CCP, which might be connected to the different catalytic activity. In particular, in KatG the proximal residues have a major role in the stability of the protein architecture because the disruption of the proximal Trp-Asp hydrogen bond by mutation weakens heme binding to the protein. On the distal side, replacing the hydrogen-acceptor carboxamide group of Asn153 by an aspartate carboxylate group or an aliphatic residue alters or disrupts the hydrogen bond with the distal His. As a consequence, the basicity of His123 is altered. The effect of mutation on Asp152 is noteworthy. Replacement of the Asp152 with Ser makes the architecture of the protein very similar to that of CCP. The Asp152 residue, which has been shown to be important in the hydrogen peroxide oxidation reaction, is expected to be hydrogen bonded to the nitrogen atom of Ile248 which is part of the KatG-specific insertion LL1, as in other KatGs. This insertion is at one edge of the heme, and connects the distal side with the proximal helices E and F, the latter carrying the proximal His ligand. We found that the distal Asp-Ile hydrogen bond is important for the stability of the heme architecture and its alteration changes markedly the proximal His-Asp hydrogen-bond interaction.

Archaeal Proteins↗
PubMed2005

Coarse-grained strategy for modeling protein stability in concentrated solutions.

We present a coarse-grained approach for modeling the thermodynamic stability of single-domain globular proteins in concentrated aqueous solutions. Our treatment derives effective protein-protein interactions from basic structural and energetic characteristics of the native and denatured states. These characteristics, along with the intrinsic (i.e., infinite dilution) thermodynamics of folding, are calculated from elementary sequence information using a heteropolymer collapse theory. We integrate this information into Reactive Canonical Monte Carlo simulations to investigate the connections between protein sequence hydrophobicity, protein-protein interactions, protein concentration, and the thermodynamic stability of the native state. The model predicts that sequence hydrophobicity can affect how protein concentration impacts native-state stability in solution. In particular, low hydrophobicity proteins are primarily stabilized by increases in protein concentration, whereas high hydrophobicity proteins exhibit richer nonmonotonic behavior. These trends appear qualitatively consistent with the available experimental data. Although factors such as pH, salt concentration, and protein charge are also important for protein stability, our analysis suggests that some of the nontrivial experimental trends may be driven by a competition between destabilizing hydrophobic protein-protein attractions and entropic crowding effects.

Binding Sites↗
PubMed2002

Phase I and pharmacokinetic study of ABI-007, a Cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel.

PURPOSE: ABI-007 is a novel Cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel. The absence of Cremophor EL may permit ABI-007 to be administered without the premedications used routinely for the prevention of hypersensitivity reactions. Furthermore, this novel formulation permits a higher paclitaxel concentration in solution and, thus, a decreased infusion volume and time. This Phase I study examines the toxicity profile, maximum tolerated dose (MTD), and pharmacokinetics of ABI-007. EXPERIMENTAL DESIGN: ABI-007 was administered in the outpatient setting, as a 30-min infusion without premedications. Doses of ABI-007 ranged from 135 (level 0) to 375 mg/m2 (level 3). Sixteen patients participated in pharmacokinetic studies. RESULTS: Nineteen patients were treated. No acute hypersensitivity reactions were observed during the infusion period. Hematological toxicity was mild and not cumulative. Dose-limiting toxicity, which occurred in 3 of 6 patients treated at level 3 (375 mg/m2), consisted of sensory neuropathy (3 patients), stomatitis (2 patients), and superficial keratopathy (2 patients). The MTD was thus determined to be 300 mg/m2 (level 2). Pharmacokinetic analyses revealed paclitaxel C(max) and area under the curve(inf) values to increase linearly over the ABI-007 dose range of 135-300 mg/m2. C(max) and area under the curve(inf) values for individual patients correlated well with toxicity. CONCLUSIONS: ABI-007 offers several features of clinical interest, including rapid infusion rate, absence of requirement for premedication, and a high paclitaxel MTD. Our results provide support for Phase II trials to determine the antitumor activity of this drug.

Adult↗
PubMed1999

Substitution of aspartic acid at beta57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303).

In class II major histocompatibility complex (MHC) proteins, residue beta57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Asp(beta)57 participates in a conserved salt bridge that bridges the alpha and beta subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Asp(beta)57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303) differing only in having aspartic acid or alanine at position beta57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alabeta57 proteins display slightly different secondary structures relative to their Aspbeta57 counterparts. A set of three peptides shows different binding affinities for DQ(alpha1*0201, beta1*0302) relative to DQ(alpha1*0201, beta1*0303). We propose that substitution of Asp(beta)57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Asp(beta)57 DQ proteins.

Alanine↗
PubMed1999

Ras enhances Myc protein stability.

Various experiments have demonstrated a collaborative action of Myc and Ras, both in normal cell growth control as well as during oncogenesis. We now show that Ras enhances the accumulation of Myc activity by stabilizing the Myc protein. Whereas Myc has a very short half-life when produced in the absence of mitogenic signals, due to degradation by the 26S proteasome, the half-life of Myc increases markedly in growth-stimulated cells. This stabilization is dependent on the Ras/Raf/MAPK pathway and is not augmented by proteasome inhibition, suggesting that Ras inhibits the proteasome-dependent degradation of Myc. We propose that one aspect of Myc-Ras collaboration is an ability of Ras to enhance the accumulation of transcriptionally active Myc protein.

Animals↗