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Results for “Protein Stability”

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PubMed1996

Protein stability: urea-induced versus guanidine-induced unfolding of metmyoglobin.

We have studied the denaturation of metmyoglobin at pH 6.0 and 25 degrees C by urea and guanidine hydrochloride (GdnHCl) which are known to unfold the protein to the same extent. It has been observed that estimates of protein stability (delta G0N-U) from urea-induced and GdnHCl-induced denaturations do not agree with one another; the linear extrapolation method gave delta G0N-U values of 7.59 +/- 0.33 and 5.35 +/- 0.10 kcal mol-1 for urea and GdnHCl denaturations, respectively. Measurements of the effect of the addition of KCl in the concentration range 0.1-1.0 M to urea denaturation have suggested that this disagreement is not due to the nonionic and ionic characters of urea and GdnHCl, respectively. The functional dependence of the free energy change of unfolding (delta GN-U) on [denaturant], the molar concentration of the denaturant, has been investigated for understanding the cause(s) of the disagreement between the two estimates of delta G0N-U of metmyoglobin. For this purpose, we have studied the GdnHCl-induced denaturation of the protein in the presence of different urea concentrations at pH 6.0 and 25 degrees C and vice versa. These measurements yield delta GN-U values in the full concentration range [Ahmad et al. (1994) J. Biochem. 115, 322-327], and these results provide strong evidence that the delta GN-U dependence on [urea] is linear (linear free energy model of denaturation) and the relation between delta GN-U and [GdnHCl] is curved (binding model of denaturation). It has been observed that the extrapolated value of delta GN-U in urea using the linear free energy model becomes identical to the extrapolated value of delta GN-U in GdnHCl using the binding model.

Animals↗

PubMed1989

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions.

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.

Animals↗

PubMed2003

How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme.

To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.

Amino Acid Substitution↗

PubMed1993

Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability.

The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.

Alanine↗

PubMed2004

Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2.

Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with normal hearts and in those with underlying disease such as heart failure. In animals with heart failure and in patients with inherited forms of exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2 (FKBP12.6) from the ryanodine receptor-calcium release channel (RyR2) complex causes an intracellular Ca2+ leak that can trigger fatal cardiac arrhythmias. A derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2 for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may be a therapeutic strategy for common ventricular arrhythmias.

Animals↗

PubMed2004

Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia.

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.

Animals↗

PubMed2002

A domain of the manganese-stabilizing protein from Synechococcus elongatus involved in functional binding to photosystem II.

Site-directed mutagenesis was performed to investigate whether the two protease-sensitive sequences Phe(156)-Gly(163) and Arg(184)-Ser(191), of the manganese-stabilizing protein (MSP) from a thermophilic cyanobacterium, Synechococcus elongatus (Motoki, A., Shimazu, T., Hirano, M., and Katoh, S. (1998) Biochim. Biophys. Acta 1365, 492-502), are involved in functional interaction with photosystem II (PSII). The ability of MSP to bind to its functional site on the PSII complex and to reactivate oxygen evolution was dramatically reduced by the substitution of Arg(152), Asp(158), Lys(160), or Arg(162) with uncharged residues, by insertion of a single residue between Phe(156) and Leu(157), or by deletion of Leu(157). Substitution of each of the four charged residues with an identically charged residue showed that the charges at Asp(158), and possibly Lys(160), are important for the electrostatic interaction with PSII. The reactivating ability was also strongly affected by the alteration of Phe(156) to Leu. Replacement of Lys(188), the only strictly conserved charged residue in the Arg(184)-Ser(191) sequence, by Gln had only a marginal effect on the function of MSP. High affinity binding of MSP to PSII was also affected significantly by mutation at Arg(152), which is located in a region (Val(148)-Arg(152)) strictly conserved among the 14 sequences so far reported. These results imply that the Val(148)-Gly(163) sequence, which is well conserved among MSPs from cyanobacteria to higher plants, is a domain of MSP for functional interaction with PSII.

Amino Acid Sequence↗

PubMed2007

Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1.

The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.

Animals↗

PubMed2004

Origins of protein stability revealed by comparing crystal structures of TATA binding proteins.

The crystal structure of TATA binding protein (TBP) from a mesothermophilic archaeon, Sulfolobus acidocaldarius, has been determined at a resolution of 2.0 A with an R factor of 20.9%. By comparing this structure with the structures of TBPs from a hyperthermophilic archaeon and mesophilic eukaryotes, as well as by comparing amino acid sequences of TBPs from archaea, covering a wide range of optimum growth temperatures, two significant determinants of the stability of TBP have been identified: increasing the interior hydrophobicity by interaction between three residues, Val, Leu, and Ile, with further differentiation of the surface, and increasing its hydrophilicity and raising the cost of unfolding. These findings suggest directions along which the stability of TBP can be engineered.

Amino Acid Sequence↗

PubMed1998

Lactate dehydrogenase from the hyperthermophilic bacterium thermotoga maritima: the crystal structure at 2.1 A resolution reveals strategies for intrinsic protein stabilization.

BACKGROUND: L(+)-Lactate dehydrogenase (LDH) catalyzes the last step in anaerobic glycolysis, the conversion of pyruvate to lactate, with the concomitant oxidation of NADH. Extensive physicochemical and structural investigations of LDHs from both mesophilic and thermophilic organisms have been undertaken in order to study the temperature adaptation of proteins. In this study we aimed to determine the high-resolution structure of LDH from the hyperthermophilic bacterium Thermotoga maritima (TmLDH), the most thermostable LDH to be isolated so far. It was hoped that the structure of TmLDH would serve as a model system to reveal strategies of protein stabilization at temperatures near the boiling point of water. RESULTS: The crystal structure of the extremely thermostable TmLDH has been determined at 2.1 A resolution as a quaternary complex with the cofactor NADH, the allosteric activator fructose-1,6-bisphosphate, and the substrate analog oxamate. The structure of TmLDH was solved by Patterson search methods using a homology-based model as a search probe. The native tetramer shows perfect 222 symmetry. Structural comparisons with five LDHs from mesophilic and moderately thermophilic organisms and with other ultrastable enzymes from T. maritima reveal possible strategies of protein thermostabilization. CONCLUSIONS: Structural analysis of TmLDH and comparison of the enzyme to moderately thermophilic and mesophilic homologs reveals a strong conservation of both the three-dimensional fold and the catalytic mechanism. Going from lower to higher physiological temperatures a variety of structural differences can be observed: an increased number of intrasubunit ion pairs; a decrease of the ratio of hydrophobic to charged surface area, mainly caused by an increased number of arginine and glutamate sidechains on the protein surface; an increased secondary structure content including an additional unique 'thermohelix' (alphaT) in TmLDH; more tightly bound intersubunit contacts mainly based on hydrophobic interactions; and a decrease in both the number and the total volume of internal cavities. Similar strategies for thermal adaptation can be observed in other enzymes from T. maritima.

Amino Acid Sequence↗

PubMed1992

Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability.

A single histidine residue has been placed at either the N-terminus or the C-terminus of each of the two alpha-helices of barnase. The pKa of that histidine residue in each of the four mutants has been determined by 1H NMR. The pKas of the two residues at the C-terminus are, on average, 0.5 unit higher, and those of the residues at the N-terminus are 0.8 unit lower, than the pKa of histidines in unfolded barnase at low ionic strength. The conformational stability of the mutant proteins at different values of pH has been measured by urea denaturation. C-Terminal histidine mutants are approximately 0.6 kcal mol-1 more stable when the introduced histidine is protonated, both at low and high ionic strength. N-Terminal mutants with a protonated histidine residue are approximately 1.1 kcal mol-1 less stable at low ionic strength and 0.5 kcal mol-1 less stable at high ionic strength (1 M NaCl). The low-field 1H NMR spectra of the mutant proteins at low pH suggest that the C-terminal histidines form hydrogen bonds with the protein while the N-terminal histidines do not form the same. The perturbations of pKa and stability result from a combination of different electrostatic environments and hydrogen-bonding patterns at either ends of helices. The value of 0.6 kcal mol-1 represents a lower limit to the favorable electrostatic interaction between the alpha-helix dipole and a protonated histidine residue at the C-terminal end of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

The phage T4 uvs Y recombination protein stabilizes presynaptic filaments.

The bacteriophage T4 uvsY protein is required for efficient recombination in T4-infected Escherichia coli cells. Previous in vitro work has shown that the purified uvsY protein is an accessory protein; it stimulates homologous pairing catalyzed by the phage uvsX protein (a RecA-like recombinase) under certain conditions. We show here that this effect can be traced, at least in part, to a UvsY-dependent stabilization of uvsX protein-single-stranded DNA complexes. These presynaptic filaments are one of the early obligatory intermediates in the strand exchange reaction between homologous single- and double-stranded DNAs. The mechanism of filament stabilization seems to involve a slower loss of UvsX subunits. A model that accounts for the data is presented in which both recombination proteins are incorporated into the presynaptic filament.

Adenosine Triphosphatases↗

PubMed2001

Optimality of the genetic code with respect to protein stability and amino-acid frequencies.

BACKGROUND: The genetic code is known to be efficient in limiting the effect of mistranslation errors. A misread codon often codes for the same amino acid or one with similar biochemical properties, so the structure and function of the coded protein remain relatively unaltered. Previous studies have attempted to address this question quantitatively, by estimating the fraction of randomly generated codes that do better than the genetic code in respect of overall robustness. We extended these results by investigating the role of amino-acid frequencies in the optimality of the genetic code. RESULTS: We found that taking the amino-acid frequency into account decreases the fraction of random codes that beat the natural code. This effect is particularly pronounced when more refined measures of the amino-acid substitution cost are used than hydrophobicity. To show this, we devised a new cost function by evaluating in silico the change in folding free energy caused by all possible point mutations in a set of protein structures. With this function, which measures protein stability while being unrelated to the code's structure, we estimated that around two random codes in a billion (109) are fitter than the natural code. When alternative codes are restricted to those that interchange biosynthetically related amino acids, the genetic code appears even more optimal. CONCLUSIONS: These results lead us to discuss the role of amino-acid frequencies and other parameters in the genetic code's evolution, in an attempt to propose a tentative picture of primitive life.

Amino Acid Substitution↗

PubMed1997

Yz. reduction kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

Catalysis↗

PubMed1991

Insertional inactivation of the psbO gene encoding the manganese stabilizing protein of photosystem II in the cyanobacterium Synechococcus PCC7942. Effect on photosynthetic water oxidation and L-amino acid oxidase activity.

A Synechococcus PCC7942 mutant in which the psbO gene was inactivated by insertion of a chloramphenicol interposon and which did not contain any detectable manganese stabilizing protein in immunoblot experiments, was constructed. Such a Synechococcus mutant was able to grow under photoautotrophic conditions. Isolated thylakoid membranes from the mutant required addition of CaCl2 and MnCl2 for photosynthetic O2 evolution, and the detectable L-amino acid oxidase activity in the isolated thylakoid membranes from the mutant was approximately four times higher than in wild-type thylakoids. The results are discussed with respect to our model suggesting that the water-oxidizing enzyme may have evolved from a flavoprotein with L-amino acid dehydrogenase/oxidase activity.

Amino Acid Oxidoreductases↗

PubMed2003

Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP.

Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP "mutant" with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 A, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.

Alanine↗

PubMed1996

The manganese stabilizing protein of photosystem II modifies the in vivo deactivation and photoactivation kinetics of the H2O oxidation complex in Synechocystis sp. PCC6803.

Dark deactivation and photoactivation of H2O-splitting activity were examined in a directed mutant (delta psbO) of Synechocystis sp. PCC6803 lacking the extrinsic manganese-stabilizing protein of the photosystem II (PSII) reaction center complex. Rapid (t1/2 = 10 min) losses of H2O-splitting activity were observed for delta psbO cells kept in the dark, but not for wild-type cells. The loss of H2O-splitting activity by delta psbO cells was suppressed by maintaining the cells under illumination and dark losses were rapidly (t1/2 < 1 min) reversed by light. Photoactivation kinetics of delta psbO and wild-type cells were compared following hydroxylamine extraction of PSII Mn. Photoactivation of delta psbO cells under continuous illumination occurs at an intrinsically faster rate (about 4-fold) than the wild-type. Virtually all of the increase in the rate of photoactivation can be accounted for by a corresponding 4-fold increase in the relative quantum yield of photoactivation as indicated by the yield of photoactivation as a function of flash number. The flash frequency dependence of photoactivation indicates a multi-quantum process in the mutant resembling the wild-type, but with significant increases in yields at all flash frequencies examined. The higher quantum yield of photoactivation in delta psbO cells occurs in the absence of large changes in the kinetics of the rate-limiting dark rearrangement. The results are consistent with increased accessibility (or affinity) and photooxidation of Mn2+ at one or both of the two binding sites involved in the initial stages of the photoactivation mechanism. In the context of previous results, it is proposed that MSP regulates the binding/photooxidation of the second Mn2+ of the photoligation sequence, but not the first.

Bacterial Proteins↗

PubMed1994

The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability.

The second vegetative sigma factor sigma S (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/sigma S expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability. rpoS transcription is inversely correlated with growth rate and is negatively controlled by cAMP-CRP. In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced. rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity. A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation. Furthermore, sigma S is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation. When cells are grown in minimal glucose medium, translational induction and sigma S stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation. Although sigma S does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level. Our analysis also indicates that at least five different signals [cAMP, a growth rate-related signal (ppGpp?), a cell density signal, an osmotic signal, and a starvation signal] are involved in the control of all these processes that regulate rpoS/sigma S expression.

Base Sequence↗