Acid Denaturation Research Articles

Characterization of the heme-histidine cross-link in cyanobacterial hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.

3P040 緑色蛍光蛋白質GFPの酸変性と巻き戻り(蛋白質 C) 物性 : 安定性、折れたたみなど)

3P040 緑色蛍光蛋白質GFPの酸変性と巻き戻り(蛋白質 C) 物性 : 安定性、折れたたみなど)

Structural properties of cyanobacterial hemoglobins: the unusual heme-protein cross-link of Synechocystis sp. PCC 6803 Hb and Synechococcus sp. PCC 7002 Hb

The truncated hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 ligate the heme iron with two axial histidines (HisF8 and HisE10). In addition, these two proteins are able to form a heme-protein cross-link between a vinyl substituent and a histidine at position 16 of the H helix. The product is a protein with improved resistance to thermal and acid denaturation.

Effect of organic mulches on soil bacterial communities one year after application

The application of organic mulches as a soil cover is effective in improving the quality of soil. However, very little information is available on the effect of mulches on the soil microbial community. In this study, we investigated the effect of various organic mulches on soil dehydrogenase activity (DHA) and microbial community structures in the top 1 cm and 5 cm below the soil surface 1 year after application of the mulches. DHA was stimulated at both depths in plots mulched with grass clippings (GC), but was not significantly different from the control for the other mulch treatments. Fatty acid methyl ester (FAME) analysis and denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rDNA fragments were used to assess changes in the soil microbial community structure. Cluster analysis and principle component analysis of FAME profiles showed that only soil mulched with pine chips distinctively clustered from the other treatments. At the soil surface, bacterial DGGE profiles revealed that distinct shifts in several bacterial populations occurred in soils mulched with GC and eucalyptus yardwaste (EY), while DGGE profiles from soil at the 5 cm depth revealed no distinct changes. Changes in bacterial diversity at the soil surface under different mulches were calculated based on the number of bands in the DGGE profile using the Shannon-Weaver index of diversity ( H). Compared to the control ( H =0.9), the GC- and EY-treated soils showed slightly increased bacterial diversity, with an H of 1.1 and 1.0, respectively. These results indicate that the long-term effect of organic mulches on the soil microbial activity and community structure is highly dependent upon the type of mulch and is mostly exerted in the top few centimeters of the soil profile.

High-resolution Structures of Retinol-binding Protein in Complex with Retinol: pH-induced Protein Structural Changes in the Crystal State

The targeted delivery of non-polar ligands by binding proteins to membranes or membrane receptors involves the release of these ligands on or near the plasma membrane of target cells. Because these hydrophobic ligands are often bound inside a deep cavity of binding proteins, as shown previously for plasma retinol-binding protein (RBP), their release from these proteins might require the destabilization of the protein structure by partially denaturing conditions, such as those possibly present near plasma membranes. RBP is a plasma transport protein which delivers specifically retinol from its store sites to target cells. Here, we report the high-resolution (1.1–1.4Å) crystal structures of bovine holo-RBP at five different pH values, ranging from 9 to 2. While unraveling details of the native protein structure and of the interactions with retinol at nearly atomic resolution at neutral pH, this study provides evidence for definite pH-induced modifications of several structural features of RBP. The structure most representative of the changes that holo-RBP undergoes at different pH values is that of its flexible state at pH 2. At this pH, most significant are the alteration of the arrangement of salt bridges and of the network of water molecules/H-bonds that participates in the retinol–RBP interaction, an appreciable increase of the volume of the β-barrel cavity, a considerably higher degree of mobility of the RBP-bound ligand and of several protein regions and the disorder of a large number of solvent molecules that are ordered at neutral pH. These changes are likely to be accompanied by a modification of the pattern of charge distribution on the protein surface. All these changes, which reveal a substantially lowered conformational stability of RBP, presumably occur at the initial stages of the acidic denaturation of RBP and are possibly associated with a facilitated release of the retinol molecule from its carrier protein.

Two-dimensional crystallization of rabbit C-reactive protein monomeric subunits.

C-reactive protein (CRP) is one of the most characteristic acute-phase proteins. Modified CRP is the monomeric form of native CRP and has recently been suggested to exist under physiological conditions. In the current work, CRP subunits were separated from stock CRP solution by size-exclusion chromatography. Two forms of two-dimensional crystals composed of monomeric CRP were obtained on negatively charged lipid monolayers: a previously reported form, MI, and a new form, MII. A projection map at 2.0 nm resolution of the two-dimensional MII crystals was obtained. The formation of the two forms of two-dimensional crystal exhibited a dependence on pH. At pH values of less than 5.5 the subunits assembled in MI packing, while at pH values greater than 6.5 they assembled in MII packing. When using modified CRP prepared by acidic denaturation, only MI crystals could be formed. The fact that CRP subunits produced by dissociation or denaturation could form highly ordered two-dimensional crystals indicates that they have a certain homogeneous structure, which supports the previous suggestion of the existence of modified CRP in vivo.

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.

Site-Specific Mutations Provide New Insights into the Origin of pH Effects and Alternative Spectral Forms in the Photoactive Yellow Protein from Halorhodospira halophila

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.

Binding of odorants to individual proteins and their mixtures. Effects of protein denaturation and association. A plasticized globule state

The unfolding-association-refolding behaviour of proteins and their interactions with odorants are significant to the processing and flavoring of foods. Interactions of 2-octanone and vanillin (aliphatic and aromatic odorants) with individual proteins and their mixtures have been studied using: native, acid and thermally denatured bovine serum albumin (BSA), native and thermally denatured ovalbumin (OA) and bovine β-casein (BC). Equilibrium dialysis and differential scanning calorimetry methods were used to study flavor binding and flavor release in protein solutions. The binding of vanillin to native BSA exhibited two equivalent binding sites with binding constants in the order of 103 M−1 and these binding parameters were not markedly changed within the range of temperature from 25 to 63 °C. The binding site number was monotonously reduced to about zero as the pH was decreased from 6.4 to 2.5, consistent with the N⇔F transition of BSA. The complete loss of vanillin binding activity was also coupled with both acid and thermal denaturation of BSA. Neither the associated BC nor thermally denatured OA produced a detectable competition with BSA for the binding of vanillin or octanone. The associated BC did not bind vanillin. It has been proposed that the adsorption of hydrophobic low-molecular weight compounds acts to plasticize the globule state of the protein. Plasticized globular proteins seem to tend towards self-association and refolding with separation of hydrophobic ligands (e.g. odorants).

緑色蛍光蛋白質変異体(Cycle3)の酸変性と巻き戻り

緑色蛍光蛋白質変異体(Cycle3)の酸変性と巻き戻り

A pH-jump approach for investigating secondary structure refolding kinetics in RNA.

It has been shown that premature translation of the plasmid-mediated toxin in hok/sok of plasmid R1 and pnd/pndB of plasmid R483 is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins at the 5'-end of the mRNA. Here, an experimental approach is presented, which allows the accurate measurement of the refolding kinetics of the 5'-end RNA fragments in vitro without chemically modifying the RNA. The method is based on acid denaturation followed by a pH-jump to neutral pH as a novel way to trap kinetically favoured RNA secondary structures, allowing the measurement of a wide range of biologically relevant refolding rates, with or without the use of standard stopped-flow equipment. The refolding rates from the metastable to the stable conformation in both the hok74 and pnd58 5'-end RNA fragments were determined by using UV absorbance changes corresponding to the structural rearrangements. The measured energy barriers showed that the refolding path does not need complete unfolding of the metastable structures before the formation of the final structures. Two alternative models of such a pathway are discussed.

In situ monitoring of gold-surface adsorption and acidic denaturation of human serum albumin by an isolation-capacitance-adopted electrochemical quartz crystal impedance system

Combined measurements of piezoelectric quartz crystal impedance (PQCI) and electrochemical impedance spectrum (EIS) using a suitable isolation capacitance is reported for the first time to monitor in situ adsorption and acidic denaturation of human serum albumin (HSA) on gold electrodes in Britton–Robinson (B–R) buffers. This method provides simultaneously mutual-interference-free and accurate parameters of EIS and PQCI. Effects of surface thiol-modification, electrode-potential and solution pH on HSA adsorption were examined and discussed. Comparative experiments of HSA adsorption in a B–R buffer of pH 6.42 on bare, cysteine- and 1-dodecanethiol-modified gold electrodes revealed that HSA adsorption is more significant on a hydrophobic (1-dodecanethiol-modified) surface. Insignificant electrode-potential effect implied minor electrostatic effects on HSA adsorption. The adsorption amount of HSA at pH 3.28 was found to be notably greater than those at pH 4.84 and 6.42. To characterize HSA adsorption, electrode standard rate constants ( k s) of the Fe(CN) 6 3−/Fe(CN) 6 4− couple were measured before and after HSA adsorption. The k s-pH curves on an HSA-modified Au electrode revealed that k s increased abruptly with the decrease of solution pH below pH ∼4. Moreover, pH-dependent responses of the resonant frequency, the motional resistance, the double-layer capacitance, the capacitance of adsorbed HSA layer and the peak absorbance of HSA solutions at 278 nm all exhibited an inflexion change at pH ∼4, and these findings have been explained on the basis of acidic denaturation of HSA and electrical charges carried by HSA molecules.

Conformation of β2-Microglobulin Amyloid Fibrils Analyzed by Reduction of the Disulfide Bond

Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.

Calorimetric study of interaction of ovalbumin with vanillin

The effect of vanillin on thermal denaturation of ovalbumin was studied using high-sensitivity differential scanning calorimetry. At neutral and acidic (6.7 and 3.0) pH values thermal denaturation of ovalbumin depends on the heating rate and is irreversible. The experimental denaturation thermograms of ovalbumin were compared with thermograms simulated according to the Lumry–Eyring model of irreversible protein denaturation (N↔D→A). It was shown that general tendencies of changes in the calorimetric parameters of ovalbumin denaturation as a function of the heating rate are in accordance with those predicted by this model. It was concluded that the heating rate could be considered as a factor shifting the denaturation equilibrium of ovalbumin (N↔D) similarly to its shifts caused by such physicochemical factors as pH, ionic strength or ligand concentration. Both the temperature and the enthalpy of ovalbumin denaturation decrease with an increase in the vanillin concentration within the pH range from 3.0 to 9.0. This effect of vanillin binding does not depend on the heating rate. The equilibrium parameters of ovalbumin denaturation have been determined by an extrapolation procedure at different vanillin concentrations. The equilibrium denaturation parameters have been used for calculation of the excess free energy of denaturation as a function of vanillin concentration. The vanillin binding constant K b equals 5 M −1 at pH 6.7 and 33 M −1 at pH 3.0. The denaturation increment of the numbers of binding sites is Δ d ν=20 at pH 6.7 and Δ d ν=3 at pH 3.0. Possible mechanism of the vanillin binding to ovalbumin and its applied aspects are discussed.

Searching for an in vivo site for nascent amyloid fibril formation.

Research in Alzheimer’s disease (AD), more than in any other amyloid-related diseases, has brought together the concerted efforts of scientists from diverse disciplines. As such, it has advanced our understanding of the in vitro molecular and biochemical parameters required for amyloid fibril formation. Evidence shows that synthetic or recombinant amyloidogenic proteins yield amyloid fibrils in vitro both in physiological and acidic conditions [6,21,23,66,73]. Importantly, addition of amyloid “nucleus” to solutions containing amyloidogenic peptides significantly reduces the lag time and accelerates fibrillogenesis. Mechanistically amyloidogenesis appears to be a nucleation-dependent process [35]. Such an event could conceivably account for relatively robust expansion of amyloid deposition in vivo and thus constitute a central event in the genesis of amyloid-related diseases. However, the question that still remains unanswered is the conditions that might affect nascent amyloid “nucleus” formation in vivo. Clearly, both for the understanding of the biology of amyloidogenesis in vivo and the development of therapeutic strategies, it is essential to understand the conditions and identify the cellular compartment(s) in which nascent amyloid fibrils are formed. Based on in vitro findings,Kelly et al. proposed “acid denaturation pathway” as a fundamental mechanism for amyloidogenesis [34,35]. Experimental evidence supports this concept, particularly the ease with which various amyloid precursor proteins or their cleaved products yield amyloid fibrils in vitro. Some of the examples are light chain of immunoglobulin, transthyretin, various fragments of the Alzheimer amyloid (Aβ) polypeptide, prion protein and serum amyloid A (SAA) etc [6, 21,23,34,35,46,66,73]. Recently, Dobson’s group introduced a more provocative concept of amyloidogenesis [11,27]. Using non-amyloidogenic proteins they were able to generate amyloid fibrils via acid denaturation pathway in vitro and propose that amyloid fibril formation may not be restricted only to certain protein sequences but a property common to many natural polypeptide chains provided that target proteins are exposed to certain “appropriate conditions”. Precise clues to the “appropriate conditions” are unclear at this time and thus a subject of intense investigation. Conceptually, acid conditions in endosomes-lysosomes (EL) in monocytoid cells, simulating the in vitro conditions, have been suggested to provide in vivo one of the requisite conditions for amyloidogenesis [11,27,34, 35]. Other accessory “appropriate conditions”, such as overloading of the EL with amyloidogenic precursor proteins, its partial degradation (although, not fully established in all cases), followed by oxidation may further augment the fibrillogenesis process [2,10,41]. Activated monocytoid cells (macrophage, MA; reticuloendothelial, RE, cells; microglia) can provide some or all of the requisite amyloid inducing conditions in vivo. Our hypothesis on the induction of inflammationassociated AA amyloidosis is that incessant intake of amyloidogenic serum amyloid A (SAA), by MA or RE cells, may cause over-loading of EL and consequently compromise their degradation activities [1,13]. As depicted in Fig. 1, reactive oxygen radicals (ROR) gener-

Binding of aroma compounds with legumin. I. Binding of hexyl acetate with 11S globulin depending on the protein molecular state in aqueous medium

We report on the role of the protein molecular state in binding and release of an aroma compound in an aqueous medium. For this purpose, the quantitative binding parameters (these are, a total number of binding sites in the protein molecule, the intrinsic binding constant and Gibbs free energy of binding) of hexyl acetate (HxAc) (the major component of a wide variety of flavours) to 11S globulin of Vicia faba (the main storage protein of broad beans) were determined in an aqueous medium by a combination of ultrafiltration and gas–liquid chromatography under different experimental conditions, namely, at pH 7.2 (the native state of 11S globulin) as well as after both the acidic (pH 3.0) and heat (30 min at 90 °C, pH 7.2) denaturation of the protein. The native legumin possessed the most binding affinity for HxAc through its unique quaternary structure. The acid denaturation of the protein led to the complete loss of the protein capacity to bind HxAc. On the heat denaturation, the protein retained its capacity to bind the aroma compound but had significantly different binding parameters and binding mechanism from those of the native protein. A complete reversibility relative to the changes in concentration of the aroma compound has been observed for binding of HxAc to the native 11S globulin as distinct from the heat denatured protein.

Cold denaturation of the hammerhead ribozyme.

Cold denaturation is a thermodynamic phenomenon resulting from a difference in the heat capacities, DeltaCp, of the folded and unfolded states of a macromolecule. Whereas this phenomenon has been extensively studied in proteins, it has been thought not to occur in nucleic acids due to a negligible DeltaCp of folding. Questioning the validity of this assumption, the low-temperature structure of the hammerhead ribozyme, a small catalytic RNA, was investigated by circular dichroism spectroscopy. In the presence of 10 mM Mg2+ at pH 5.0 and 40% methanol, a cold unfolding event likely corresponding to tertiary structure loss was observed with a Tm of -20 degrees C. In 500 mM NaCl at pH 6.6, and 40% methanol, large-scale unfolding of the ribozyme at both hot (Tm = 53 degrees C) and cold (Tm = -1 degrees C) temperatures occurred. Fitting of these data to a two-state model allowed determination of DeltaCp = 3.4 kJ mol-1 K-1, corresponding to >/=0.18 kJ K-1 (mol base pair)-1, in good agreement with recently published calorimetric values for DNA duplexes. These results constitute the first direct observation of cold denaturation of a nucleic acid, and point to the importance of DeltaCp terms in the thermodynamics of nucleic acid folding.

1I0945 緑色蛍光蛋白質変異体(Cycle3)の酸変性と巻き戻り(1.蛋白質(C)物性,一般演題,日本生物物理学会第40回年会)

1I0945 緑色蛍光蛋白質変異体(Cycle3)の酸変性と巻き戻り(1.蛋白質(C)物性,一般演題,日本生物物理学会第40回年会)

Global configuration of single titin molecules observed through chain-associated rhodamine dimers.

The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding/refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.