Stability Of Apoprotein Research Articles

Effect of Tb(III) on the unfolding of ciliate Euplotes octocarinatus centrin induced by guanidine hydrochloride

To understand the unfolding of ciliate Euplotes octocarinatus centrin (EoCen), the glycine positioned at 115, the sixth residue of the loop of the protein's third EF-hand, was mutated into tryptophan (Trp). Intrinsic fluorescence and Tb(III) binding properties of wild type EoCen and G115W mutant were monitored by fluorescence spectra in 10 mmol/L Hepes. The emission maximum of EoCen was 306 nm and mutation had no impact on the Tb(III) binding properties. The properties of G115W were investigated by fluorescence, far-UV circular dichroism (CD) spectra and fluorescence decays in the absence or in the presence of 6 mol/L guanidine hydrochloride (GdnHCl). For the increase in polarity of micro-environment around Trp residue, the emission maximum of apoG115W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCl. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCl. The unfolding of G115W induced by GdnHCl was assessed by using the model of structural element. The unfolding of proteins is a sequential reaction, namely two-transition, three-state process. The first transition belongs to the unfolding of the C-terminal domain, and the second transition is assigned to the unfolding of the N-terminal domain. The Δ〈ΔGtotal0(H2O)〉 was used to determine the effect of Tb(III) on the stability of apoprotein. The 〈ΔGtotal0(H2O)〉 for Tb2-G115W has a less increase of 0.68 kJ/mol compared with apoG115W, proving Tb(III) situated at C-terminal has negligible impact on the stability of protein. Whereas the 〈ΔGtotal0(H2O)〉 for Tb4-G115W has a rise of 1.29 kJ/mol compared with Tb2-G115W, manifesting Tb(III) located at low affinity sites has considerable influence on protein stability, mainly stabilizing the N-terminal domain.

Structural changes induced by acidic pH in human apolipoprotein B-100.

Acidification in the endosome causes lipoprotein release by promoting a conformational change in the LDLR allowing its recycling and degradation of LDL. Notwithstanding conformational changes occurring in the LDLR have expanded considerably, structural changes occurring in LDL particles have not been fully explored yet. The objectives of the present work were to study structural changes occurring in apoB100 by infrared spectroscopy (IR) and also LDL size and morphology by dynamic light scattering (DLS) and electron microscopy (EM) at both pH 7.4 and 5.0. We determined by IR that pH acidification from 7.4 to 5.0, resembling that occurring within endosomal environment, induces a huge reversible structural rearrangement of apoB100 that is characterized by a reduction of beta-sheet content in favor of alpha-helix structures. Data obtained from DLS and EM showed no appreciable differences in size and morphology of LDL. These structural changes observed in apoB100, which are likely implied in particle release from lipoprotein receptor, also compromise the apoprotein stability what would facilitate LDL degradation. In conclusion, the obtained results reveal a more dynamic picture of the LDL/LDLR dissociation process than previously perceived and provide new structural insights into LDL/LDLR interactions than can occur at endosomal low-pH milieu.

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

Comparison of cytochromes b5 from insects and vertebrates

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

Low-Affinity Signature of the Rat β-Parvalbumin CD Site. Evidence for Remote Determinants

Although rat beta-parvalbumin and chicken parvalbumin 3 (CPV3) are identical at 74 of 108 residues, rat beta exhibits perceptibly lower Ca2+ and Mg2+ affinities. At 25 degrees C, in Hepes-buffered saline, at pH 7.4, the overall deltadeltaG degrees ' values are 2.0 and 3.9 kcal/mol, respectively. These differences primarily reflect the disparate behavior of the CD sites in the two proteins. Their respective binding constants for Ca2+, for example, are 1.5 x 10(6) and 2.4 x 10(7) M-1. The extent to which this differential behavior is dictated by local and remote sequence differences is unknown. To explore this question, we performed mutagenesis on rat beta, substituting the corresponding CPV3 codon for residues 49, 50, 57, 58, 59, and 60. The resulting CD site is identical to CPV3 at 27 of 30 positions. The mutations were introduced in four stages, replacing residues 49 and 50 (yielding beta 49/50), then 57 and 58 (beta 49/50/57/58), then 59 (beta 49/50/57/58/59), and finally 60 (beta 49/50/57/58/59/60). Apoprotein stability was examined by scanning calorimetry and chemical denaturation and divalent ion affinity by titration calorimetry. All four variants exhibit elevated Tm values and are between 0.13 and 0.39 kcal/mol more stable at 25 degrees C. Although all four proteins display heightened divalent ion affinity, the increases are small. The maximal deltadeltaG degrees ' values, observed for 49/50/57/58/59/60, are just -0.56 and -0.96 kcal/mol for Ca2+ and Mg2+, respectively. Evidently, structural features beyond the metal ion-binding motif contribute to the unusual divalent ion-binding behavior associated with the rat beta CD site.

Divergence in Nonspecific Hydrophobic Packing Interactions in the Apo State, and Its Possible Role in Functional Specialization of Mitochondrial and Microsomal Cytochromeb5

The outer mitochondrial membrane isoform of mammalian cytochrome b(5) (OM b(5)) is distinguished from the microsomal isoform (Mc b(5)) by its considerably greater stability. In contrast, OM and Mc apocytochrome b(5) (apo-b(5)) exhibit similar thermodynamic stability. Contributing substantially to the greater stability of OM b(5) relative to that of Mc b(5) is the presence of Leu at position 71. Replacing Leu-71 in OM b(5) with the corresponding Mc b(5) residue (Ser) not only diminishes holoprotein stability but also markedly compromises apoprotein stability. The studies reported herein were undertaken to clarify the role played by Leu-71 in stabilizing OM b(5)s relative to Mc b(5)s, and were motivated by the possibility that stability is related to other differences in OM and Mc b(5) properties that are important for their specialized subcellular roles. The results of these studies show that Leu-71 plays an essential role in maintaining the structural integrity of the heme-independent folding core of OM apo-b(5) (core 2), despite its location in the disordered empty heme-binding pocket (core 1). The conformational integrity of core 2 in Mc apo-b(5)s is not similarly dependent on the presence of a hydrophobic residue at position 71, providing new evidence for evolution of compensating structural features not present in OM b(5)s. We propose that Leu-71 achieves its effect on OM apo-b(5) core 2 structure by participating in a nonspecific hydrophobic collapse of disordered core 1, templated by more conformationally restricted side chains of residues in the beta-sheet that separates the two cores. We hypothesize that this has the added effect of maintaining core 1 of OM apo-b(5)s in a state more compact than that which occurs in Mc apo-b(5)s, possibly contributing to stronger heme binding by limiting the number of non-native conformations that the empty heme-binding pocket can populate.

A relationship between heme binding and protein stability in cytochrome b5.

Conformational changes and long-range effects are often observed in proteins when they associate with their ligands. In many cases, these structural perturbations are essential to function, and they are the result of complex networks of interactions. Here we used cytochrome b(5), a protein that undergoes extensive structural rearrangement upon heme binding, to seek a relationship between affinity for the cofactor and extent of refolding induced by its binding. Three variants of the water-soluble domain of the rat microsomal protein were chosen to affect the stability of the apoprotein or the holoprotein. Sequence alterations were introduced in the heme binding loop (type I mutations, D60R and (55)TENFED --> (55)TEPFEED, or PE), which is largely unstructured in the apoprotein state, and in the folded core of the apoprotein (type II mutation, P81A). Thermal and chemical denaturation experiments and heme transfer experiments were performed on these proteins. Type I mutations left the thermodynamic stability of the apoprotein unchanged. The first mutation (D60R) stabilized the holoprotein in a probable manifestation of enhanced helical propensity or improved electrostatic interactions. The second mutation (PE) decreased heme affinity and holoprotein stability in concert. For this protein, heme transfer experiments could be used to estimate the rate constant of heme loss from each of the heme orientational isomers. In contrast, the type II mutation resulted in a marked destabilization of the apoprotein but an intermediate effect on the holoprotein stability and heme affinity. These data supported that heme affinity could be modulated by the apoprotein stability and by specific residues remote from the heme binding site.

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll proteins P700 and D1 by increasing apoprotein stability.

Chlorophyll protein accumulation in barley (Hordeum vulgare L.) chloroplasts is controlled posttranscriptionally by light-induced formation of chlorophyll a. The abundance of translation initiation complexes associated with psbA, psaA, and rbcL mRNAs was measured using extension and inhibition analysis in plants grown in the dark for 4.5 d and then illuminated for up to 16 h. Light-induced accumulation of the chlorophyll proteins was not accompanied by changes in the abundance of translation initiation complexes, indicating that regulation of chlorophyll protein accumulation at this stage of development does not occur at the level of translation initiation. Translational runoff assays were performed in the presence of lincomycin, an inhibitor of translation initiation, to determine whether chlorophyll protein accumulation was regulated at the level of translation elongation. The extent of ribosome runoff of psaA and psbA mRNAs was similar in the presence or absence of chlorophyll, indicating that chlorophyll did not alter chlorophyll protein translation elongation. Polysome-associated D1 translation intermediates were radiolabeled in the presence or absence of chlorophyll, even though full-length D1 accumulated only in the presence of chlorophyll. Chlorophyll influenced the stability of D1 translation intermediates to a small extent and greatly increased D1 stability after release from ribosomes. Overall, these results demonstrate that light-induced chlorophyll biosynthesis triggers the accumulation of the chlorophyll proteins D1 and P700 in barley chloroplasts by enhancement of chlorophyll apoprotein stability.

Synthesis of chlorophyll a regulates translation of chlorophyll a apoproteins P700, CP47, CP43 and D2 in barley etioplasts.

Accumulation of plastid-encoded chlorophyll apoproteins and chlorophyll synthesis are controlled by light in angiosperms. An in vitro system utilizing isolated and lysed barley (Hordeum vulgare L.) etioplasts revealed the specific accumulation of P700, CP47, CP43 and D2 triggered by de novo synthesis of chlorophyll. Accumulation rates of radiolabelled chlorophyll apoproteins were linear for about 30 min. Pulse/chase translation assays showed that synthesis of chlorophyll does not result in increased chlorophyll apoprotein stability. Instead turnover rates of chlorophyll apoproteins were higher in the presence than in the absence of chlorophyll. Chlorophyll-dependent accumulation of chlorophyll apoproteins must therefore be regulated on the level of translation. Translation of chlorophyll apoproteins was blocked to about 50% by addition of 30-50 microM aurintricarboxylic acid or 20 microM kasugamycin. The kinetics of chlorophyll-dependent translation indicated that the in vitro translation system is capable of translation initiation. The capability of translation initiation was lost in lysed etioplasts after preincubation for at least 5 min without chlorophyll synthesis. The results suggest that initiation is involved in chlorophyll-dependent regulation of translation.

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll apoproteins CP43 and D1 by increasing apoprotein stability.

Chlorophyll apoprotein accumulation in higher plant chloroplasts is controlled by light-dependent chlorophyll formation. Dark-grown plants lack chlorophyll and chlorophyll apoproteins. However, the plastid genes encoding the chlorophyll apoproteins are transcribed; chlorophyll apoprotein mRNA accumulates and associates with polysomes in plastids of dark-grown plants. Pulse-labeling assays revealed a population of short-lived proteins in plastids of dark-grown plants. One of these transiently labeled proteins was CP43, a chlorophyll apoprotein associated with photosystem II. Pulse-chase assays showed that newly synthesized CP43 was rapidly degraded in plastids of dark-grown plants, which lack chlorophyll. In contrast, CP43 synthesized in plastids from illuminated plants was stable. The synthesis of D1, a chlorophyll apoprotein of the photosystem II reaction center, was also analyzed in plastids of dark-grown and illuminated plants. Radiolabel accumulation into full-length D1 was only detected in plastids of illuminated plants. However, D1 translation intermediates of 15-25 kDa were detected in both plastid populations. Pulse-chase assays showed that the 15- to 25-kDa D1 translation products were precursors of mature D1 in plastids of illuminated plants. In contrast, in plastids of dark-grown plants, the 15- to 25-kDa translation intermediates were converted into a 23-kDa polypeptide previously suggested to be a proteolytic product of D1. These results indicate that chlorophyll produced in illuminated plants stabilizes D1 nascent polypeptides, which allows accumulation of mature D1.

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, two-state thermal denaturation (midpoint temperature 54 degrees). The enthalpy (2.4 cal/g)(10.0 J/g) and specific heat change (0.08 cal/degrees C per g)(0.33 J/degrees C per g) associated with the denaturation were used to calculate the free energy difference (deltaG) between native and unfolded apoA-I at 37 degrees. DeltaG (2.4 kcal/mol)(10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol)(33-59 kJ/mol), indicating that at 37 degrees native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipid in its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 85 degrees not present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, suggesting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be especially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.