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A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization.

The identification of genetic mutations responsible for rare familial forms of Parkinson's disease (PD) have provided tremendous insight into the molecular pathogenesis of this disorder. Mutations in the DJ-1 gene cause autosomal recessive early onset PD in two European families. A Dutch kindred displays a large homozygous genomic deletion encompassing exons 1-5 of the DJ-1 gene, whereas an Italian kindred harbors a single homozygous L166P missense mutation. A homozygous M26I missense mutation was also recently reported in an Ashkenazi Jewish patient with early onset PD. Mutations in DJ-1 are predicted to be loss of function. The recent determination of the crystal structure of human DJ-1 demonstrates that it exists in a homo-dimeric form in vitro, whereas the L166P mutant exists only as a monomer. Here, we examine the in vivo effects of the pathogenic L166P and M26I mutations on the properties of DJ-1 in cell culture. We report that the L166P mutation confers markedly reduced protein stability to DJ-1, which results from enhanced degradation by the 20S/26S proteasome but not from a loss of mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact to form homo-oligomers. In contrast, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-oligomers. These properties of the L166P mutation may contribute to the loss of normal DJ-1 function and are likely to be the underlying cause of early onset PD in affected members of the Italian kindred.

Autoantigens↗

Competing solvent effects of polyols and guanidine hydrochloride on protein stability.

The denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25 degrees C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. These results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.

Animals↗

Urea effects on protein stability: hydrogen bonding and the hydrophobic effect.

The effects of urea on protein stability have been studied using a model system in which we have determined the energetics of dissolution of a homologous series of cyclic dipeptides into aqueous urea solutions of varying concentration at 25 degrees C using calorimetry. The data support a model in which urea denatures proteins by decreasing the hydrophobic effect and by directly binding to the amide units via hydrogen bonds. The data indicate also that the enthalpy of amide hydrogen bond formation in water is considerably higher than previously estimated. Previous estimates included the contribution of hydrophobic transfer of the alpha-carbon resulting in an overestimate of the binding between urea and the amide unit of the backbone and an underestimate of the binding enthalpy.

Amides↗

The contribution of C alpha-H...O hydrogen bonds to membrane protein stability depends on the position of the amide.

Structural analyses of membrane proteins reveal a large number of C(alpha)-H...O contacts between transmembrane helices, presumed to be hydrogen bonds. Recent experiments produced conflicting results for the contribution of such hydrogen bonds to membrane protein stability. An FTIR study estimated an energy of -0.88 kcal/mol for the G79-C(alpha)-H...I76-O hydrogen bond in glycophorin A, whereas a mutagenesis study showed that the A51-C(alpha)-H...T24-O(gamma) hydrogen bond does not stabilize bacteriorhodopsin. Here, we reconcile these results using molecular mechanics calculations and an implicit membrane model (IMM1). With explicit hydrogen atoms, the potential energy of the G79-C(alpha)-H...I76-O interaction in GpA ranges from -0.54 to -0.9 kcal/mol and its contribution to stability (effective energy) from -0.49 to -0.83 kcal/mol, depending on the structural model used. The average values of these quantities in GpA-like motifs are similar. In bR, the corresponding numbers for the A51-C(alpha)-H...T24-O(gamma) interaction are +0.15 and +0.32 kcal/mol. The difference results from the different arrangement of the interacting groups and specifically the position of the acceptor with respect to the C(alpha) and N atoms. This conclusion likely applies to soluble proteins as well.

Alanine↗

Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes.

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.

Amino Acid Sequence↗

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration.

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.

Animals↗

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures.

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.

Aldose-Ketose Isomerases↗

Assembly and function of the photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior.

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404-414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S-S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta 1274: 135-142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the pI of the MSPs and BSA enhanced their aggregation resistance, a behavior which can be predicted from their charge (MSP) or a combination of charge and stabilization by -S-S- bonds (BSA). In the case of aggregation resistance by MSP, this is likely to be an important factor in its ability to avoid unproductive self-association reactions in favor of formation of the protein-protein interactions that lead to formation of the functional oxygen evolving complex.

Animals↗

Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine.

Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, beta-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte beta-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3M it increased the melting temperature of RNase A (Tm) by more than 12K and gave rise to a stability increase of 10.6kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (deltaC(p)) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3M. Also, here deltaCp was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by beta-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature.

Amino Acids, Diamino↗

Protein stability and dynamics in the pressure-temperature plane.

The pressure-temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.

Animals↗

Is thermostability of glucose-6-phosphatase indeed dependent on a stabilizing protein?

Partial purification of glucose-6-phosphatase from rat liver microsomes by solubilization of the membranes with the non-ionic detergent Triton X-114 at pH 6.5 and the removal of inactivating detergent by hydrophobic chromatography results in a thermostable enzyme protein which is not dependent on stabilizing phospholipids or proteins. The readdition of low amounts of detergent immediately causes a conversion into a thermo-unstable phosphohydrolase protein. Thus these findings present evidence that heat instability of partially purified glucose-6-phosphatase derives from traces of inactivating detergent changing the structural properties of the phosphohydrolase rather than from the absence of the postulated specific stabilizing protein.

Animals↗

Using protein design to dissect the effect of charged residues on metal binding and protein stability.

Ca2+ controls biological processes by interacting with proteins with different affinities, which are largely influenced by the electrostatic interaction from the local negatively charged ligand residues in the coordination sphere. We have developed a general strategy for rationally designing stable Ca2+- and Ln3+-binding proteins that retain the native folding of the host protein. Domain 1 of cluster differentiation 2 (CD2) is the host for the two designed proteins in this study. We investigate the effect of local charge on Ca2+-binding affinity based on the folding properties and metal-binding affinities of the two proteins that have similarly located Ca2+-binding sites with two shared ligand positions. While mutation and Ca2+ binding do not alter the native structure of the protein, Ca2+ binding specifically induced changes around the designed Ca2+-binding site. The designed protein with a -5 charge at the binding sphere displays a 14-, 20-, and 12-fold increase in the binding affinity for Ca2+, Tb3+, and La3+, respectively, compared to the designed protein with a -3 charge, which suggests that higher local charges are preferred for both Ca2+ and Ln3+ binding. The localized charged residues significantly decrease the thermal stability of the designed protein with a -5 charge, which has a T(m) of 41 degrees C. Wild-type CD2 has a T(m) of 61 degrees C, which is similar to the designed protein with a -3 charge. This decrease is partially restored by Ca2+ binding. The effect on the protein stability is modulated by the environment and the secondary structure locations of the charged mutations. Our study demonstrates the capability and power of protein design in unveiling key determinants to Ca2+-binding affinity without the complexities of the global conformational changes, cooperativity, and multibinding process found in most natural Ca2+-binding proteins.

Calcium↗

Protein-stabilized foams and emulsions.

This review is a survey of studies on protein-stabilized foams and emulsions in relatively simple, well-defined systems (rather than in food products per se). The emphasis is on the extent of basic understanding developed, particularly in terms of the physicochemical properties involved. The stability of the system, its formation, and its rheology are covered. Unfortunately, much work is of limited fundamental value because of poorly designed experimental approaches and the failure to measure key parameters. Reasonable generalization can be made concerning the effects of a number of variables, particularly protein solubility, pH, protein structure and the presence of small molecule surfactants. However, several possible explanations of these effects in terms of surface properties remain tenable. A number of observations can fairly confidently be ascribed to the influence of rheology on flow processes within foams and emulsions.

Emulsions↗

Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.

The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗

Threading structural model of the manganese-stabilizing protein PsbO reveals presence of two possible beta-sandwich domains.

The manganese-stabilizing protein (PsbO) is an essential component of photosystem II (PSII) and is present in all oxyphotosynthetic organisms. PsbO allows correct water splitting and oxygen evolution by stabilizing the reactions driven by the manganese cluster. Despite its important role, its structure and detailed functional mechanism are still unknown. In this article we propose a structural model based on fold recognition and molecular modeling. This model has additional support from a study of the distribution of characteristics of the PsbO sequence family, such as the distribution of conserved, apolar, tree-determinants, and correlated positions. Our threading results consistently showed PsbO as an all-beta (beta) protein, with two homologous beta domains of approximately 120 amino acids linked by a flexible Proline-Glycine-Glycine (PGG) motif. These features are compatible with a general elongated and flexible architecture, in which the two domains form a sandwich-type structure with Greek key topology. The first domain is predicted to include 8 to 9 beta-strands, the second domain 6 to 7 beta-strands. An Ig-like beta-sandwich structure was selected as a template to build the 3-D model. The second domain has, between the strands, long-loops rich in Pro and Gly that are difficult to model. One of these long loops includes a highly conserved region (between P148 and P174) and a short alpha-helix (between E181 and N188)). These regions are characteristic parts of PsbO and show that the second domain is not so similar to the template. Overall, the model was able to account for much of the experimental data reported by several authors, and it would allow the detection of key residues and regions that are proposed in this article as essential for the structure and function of PsbO.

Algorithms↗

Structural and functional differentiation of three groups of tyrosine residues by acetylation of N-acetylimidazole in manganese stabilizing protein.

To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually approximately 5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of approximately 6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.

Acetylation↗

Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation.

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.

Amino Acid Sequence↗

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding.

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.

Alanine↗