Thermal Destabilization Research Articles

ヒトMAPキナーゼJNK1の立体構造・機能・安定性に対する遊離システイン残基の置換効果

To characterize the role of cysteine residues on the structure, function and stability of JNK1, we prepared and evaluated the wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The solvent exposed cysteine residues did not influence biological function and mutating these residues raised the thermal stability because of newly formed hydrogen bonds and of higher hydration as speculated by the mutant structures. The surface cysteine involved in the molecular-surface hydrophobic pocket did not affect biological function; although a moderate thermal destabilization was observed. Cysteines in the loosely-assembled hydrophobic environment moderately contributed to thermal stability and the mutations of these cysteines had negligible effect on enzyme activity. The other cysteines are involved in the tightly-filled hydrophobic core and mutation of these residues conferred the adverse effects on the thermal stability and enzyme activity.

Polymorphism of human telomeric quadruplex structure controlled by DNA concentration: a Raman study.

DNA concentration has been recently suggested to be the reason why different arrangements are revealed for K+-stabilized human telomere quadruplexes by experimental methods requiring DNA concentrations differing by orders of magnitude. As Raman spectroscopy can be applied to DNA samples ranging from those accessible by absorption and CD spectroscopies up to extremely concentrated solutions, gels and even crystals; it has been used here to clarify polymorphism of a core human telomeric sequence G3(TTAG3)3 in the presence of K+ and Na+ ions throughout wide range of DNA concentrations. We demonstrate that the K+-structure of G3(TTAG3)3 at low DNA concentration is close to the antiparallel fold of Na+-stabilized quadruplex. On the increase of G3(TTAG3)3 concentration, a gradual transition from antiparallel to intramolecular parallel arrangement was observed, but only for thermodynamically equilibrated K+-stabilized samples. The transition is synergically supported by increased K+ concentration. However, even for extremely high G3(TTAG3)3 and K+ concentrations, an intramolecular antiparallel quadruplex is spontaneously formed from desalted non-quadruplex single-strand after addition of K+ ions. Thermal destabilization or long dwell time are necessary to induce interquadruplex transition. On the contrary, Na+-stabilized G3(TTAG3)3 retains its antiparallel folding regardless of the extremely high DNA and/or Na+ concentrations, thermal destabilization or annealing.

Seven Cysteine-Deficient Mutants Depict the Interplay between Thermal and Chemical Stabilities of Individual Cysteine Residues in Mitogen-Activated Protein Kinase c-Jun N-Terminal Kinase 1

Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.

Analysis of the phospholipase C-δ1 pleckstrin homology domain using native polyacrylamide gel electrophoresis

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP3) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP3 binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP3 and in thermal destabilization of the IP3-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP3-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP3 and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP3-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.

Structure and Stability of Duplex DNA Containing (5′S)-5′,8-Cyclo-2′-deoxyadenosine: An Oxidatively Generated Lesion Repaired by NER

Cellular respiration and ionizing radiation generate 5',8-cyclo-2'-deoxyribonucleosides, a special type of DNA damage that involves two modifications in the same nucleotide. These lesions evade the action of base excision glycosylases, and their removal is a function of the nucleotide excision repair pathway. Diastereomeric 5',8-cyclo-2'-deoxyadenosine blocks mammalian DNA replication, diminishes the levels of DNA transcription, and induces transcriptional mutagenesis. Using solution state NMR spectroscopy and restrained molecular dynamics simulations, we have determined the structure of an undecameric DNA duplex having a centrally located (5'S)-5',8-cyclo-2'-deoxyadenosine residue paired to T. The damaged duplex structure is a right-handed helix having Watson-Crick base-pair alignments throughout, and 2-deoxyribose puckers within the B-form conformation. Only small structural perturbations are observed at the lesion-containing and 5'-flanking base pair. The 2-deoxyribose of the damaged nucleotide adopts the O4'-exo conformation, and the S-cdA·T base pair is propeller twisted. The 5'-lesion-flanking base is tilted forming a significantly buckled base pair with its partner guanine. Analysis of UV-melting curves indicates mild thermal and thermodynamic destabilization on the damaged duplex. The S-cdA·T duplex structure shows many similarities to and some intriguing differences from the recently reported structure of an S-cdG·dC duplex³¹ that suggest different lesion site dynamics.

Temporal behavior of DNA thermal stability in the presence of platinum compounds. Role of monofunctional and bifunctional adducts

Penetrating into cell nuclei, antitumor drug cisplatin sequentially forms various intermediate and final adducts destroying local DNA structure. The demonstrated disappearance of the fine structure of melting curve of long DNAs along with a strong decrease in melting enthalpy conforms to the structural impact. However, the negative thermal effect (δTm) caused by cisplatin is relatively small if neutral medium is used in melting experiments. Cisplatin's inactive analogs transplatin and diethylenetriaminechloroplatinum {Pt[(dien)Cl]Cl} also distort DNA structure but their thermal effect is even positive. We have found that the use of alkaline medium in melting experiments strengthens the negative thermal effect for cisplatin. For transplatin and Pt[(dien)Cl]Cl, the thermal effect becomes negative that makes it qualitatively consistent with structural distortions. Those changes are explained by elimination of nonspecific electrostatic stabilization of DNA under platination. Additionally, alkaline medium fixes intermediate states of DNA platination and makes them stable against heating. These results allowed us to monitor δTm under binding of platinum compounds to DNA and their further transformation. The kinetic and thermal characteristics of monofunctional and bifunctional adducts were evaluated. It has been demonstrated that monofunctional adducts of cisplatin, transplatin and Pt[(dien)Cl]Cl produce approximately the same thermal destabilization. Cisplatin intrastrand crosslinks cause a two-fold stronger thermal destabilization than its monofunctional adducts. The value of δTm for cisplatin's final adducts is ten times larger than for transplatin. This difference mainly comes from the much stronger thermal destabilizing power of cisplatin's intrastrand crosslinks, which are responsible for antitumor activity of this compound.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Thermodynamic Consequences of the Hyperoxidized Guanine Lesion Guanidinohydantoin in Duplex DNA

Guanidinohydantoin (Gh) is a hyperoxidized DNA lesion produced by oxidation of 8-oxo-7,8-dihydroguanine (8-oxoG). Previous work has shown that Gh is potently mutagenic in both in vitro and in vivo coding for G → T and G → C transversion mutations. In this work, analysis by circular dichroism shows that the Gh lesion does not significantly alter the global structure of a 15-mer duplex and that the DNA remains in the B-form. However, we find that Gh causes a large decrease in the thermal stability, decreasing the duplex melting temperature by ~17 °C relative to an unmodified duplex control. Using optical melting analysis and differential scanning calorimetry, the thermodynamic parameters describing duplex melting were also determined. We find that the Gh lesion causes a dramatic decrease in the enthalpic stability of the duplex. This enthalpic destabilization is somewhat tempered by entropic stabilization; yet, Gh results in an overall decrease in thermodynamic stability of the duplex relative to a control that lacks DNA damage, with a ΔΔG° of -7 kcal/mol. These results contribute to our understanding of the consequences of hyperoxidation of G and provide insight into how the thermal and thermodynamic destabilization caused by Gh may influence replication and/or repair of the lesion.

Environment Controlled Dewetting of Rh–Pd Bilayers: A Route for Core–Shell Nanostructure Synthesis

Chemical environment plays a significant role on the size, shape, or surface composition of nanostructures. Here, the chemical environment effects are studied in the context of core–shell nanoparticle synthesis. The environment driven dynamics and kinetics of Rh/Pd bilayers is investigated by in situ ambient pressure X-ray photoelectron spectroscopy. Thin Rh (∼1.5 nm)/Pd (∼ 1.5 nm) bilayers were grown on thermally oxidized Si substrates. The films were heated in CO or NO environments or heated in vacuum with a subsequent NO/CO cycling. This study demonstrates that not the initial stacking sequence but the chemical environment plays a crucial role in controlling the surface composition. Heating in CO results in a surface enrichment of Pd at ∼200 °C and is followed by film dewetting at ∼300 °C. Heating in NO results in progressive oxidation of Rh starting at ∼150 °C, which stabilizes the film continuity up to >∼375 °C. The film rupture correlates with the thermal destabilization of the surface oxide. Heatin...

Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

Ryanodine Receptors (RyRs) are huge Ca²⁺ release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

Relative-thickness dependence of exchange bias in bilayers and trilayers

We consider the models of ferromagnetic (FM)/antiferromagnetic (AFM) bilayers and trilayers and perform a modified Monte Carlo method to study their exchange bias (EB) properties at low temperature after field cooling on increasing one component thickness at the expense of the other one. The results indicate that EB is insensitive to the thickness variations as the FM layer is thicker than the AFM one. Otherwise, it has a steep increase with the decrease of FM thickness, but the purely inverse proportion is no longer valid due to the dual influences of FM and AFM thicknesses. EB in trilayers should be approximately twice larger than that in bilayers because there is a double interfacial area in the trilayers compared with the bilayers, but the dispersed FM/AFM distributions may break this relation as a result of thermal destabilization. Moreover, EB is independent of FM/AFM stacking sequences probably because of the ideal interface between them. It has been clarified unambiguously that such control of EB through varying the FM/AFM dimensions in heterostructures is attractive for spintronics applications.

Increased PARP-1 levels in nuclear matrix isolated from heat shock treated rat liver

Poly(ADP-ribose) polymerase-1 (PARP-1), a chromatin-associated enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers onto a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. PARP-1 levels in the nuclear matrix vary depending on the matrix isolation method used. The nuclear matrix appears to be the most thermosensitive nuclear structure during heat shock. Here we provide evidence for the extensive translocation of PARP-1 from chromatin to the nuclear matrix during heat shock. This translocation is accompanied by inhibition of PARP activity in the nucleus and elevation of PARP activity in the nuclear matrix. Our data suggest that thermal destabilization of the nuclear matrix is less likely to contribute to the translocation of PARP-1 to the nuclear matrix.

Protein Disorder and Degradation: Is Ubiquitin the Missing Link?

The ubiquitin-proteasome system is responsible for degradation of numerous proteins. How the proteasome successfully degrades such a large variety of proteins is not well understood. It is thought that in order to be degraded, proteins must have a disordered region (an unstructured stretch of residues of a certain length) that can be used by the proteasome to initiate degradation. We have assembled a dataset of proteins that undergo ubiquitination and degradation, and characterized their structural properties (Hagai et al., JMB, 2011). Surprisingly, we observed that ∼25% of the proteins that are successfully degraded lack the needed disordered region. Therefore, an additional mechanism should somehow provide a disordered region to enable degradation. Since the majority of proteins are ubiquitinated prior to their degradation, it is possible that the ubiquitin chain itself can influence the structure and thermal stability of the protein.Using molecular dynamics simulations we showed that ubiquitination can significantly alter the protein's structure and stability (Hagai and Levy, PNAS, 2010). Ubiquitination of the substrate Ubc7 at the residues that are modified in vivo prior to degradation uniquely results in significant thermal destabilization and a local distortion near the modification site. These effects are specific to these sites, while other lysine residues which are not used in vivo, display diverse behavior upon ubiquitination. This indicates that ubiquitination can facilitate the unfolding process and create disordered regions, and therefore can assist degradation.Our findings suggest that in addition to its signaling role, ubiquitination may alter protein biophysics to support degradation.

A new method to synthesize very active and stable supported metal Pt catalysts: thermo-destabilization of microemulsions

A new technique to deposit nanoparticles synthesized in reverse micellar microemulsions onto support material without agglomeration is named thermal destabilization of microemulsion. The multifaceted Pt crystals, mostly truncated octahedra, were produced inside reverse micelles with an average size of 2.5 nm and a narrow size distribution. After deposition, the Pt crystals were found to be well dispersed on the support with an average size of 2.5 nm. After testing with hydrogenation of α-methyl styrene, the produced Pt-catalyst showed higher activity (6 times higher) and stability than commercial ones. The advantages of this synthesis route of nanoparticles include simple operation, and the ease of controlling the size and shape of nanoparticles without using capping agents.

Natural phenylalanine hydroxylase variants that confer a mild phenotype affect the enzyme's conformational stability and oligomerization equilibrium

Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase ( PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect of BH4 addition directly in living human cells expressing wild-type PAH or p.A403V, a mild mutant associated with BH4 responsiveness in vivo. Our results demonstrate that BH4 addition promotes re-establishment of the oligomerization equilibrium, thus indicating that the dimer-to-tetramer shift in pA403V plays a key role in BH4 responsiveness. In conclusion, we show that the oligomerization process and conformational stability are altered by mutations that could affect the physiological behavior of the enzyme. This endorses the hypothesis that oligomerization and folding defects of PAH variants are the most common causes of HPAs, particularly as regards mild human phenotypes.

DNA condensation and its thermal stability influenced by phospholipid bilayer and divalent cations

We studied the effect of divalent alkaline earth metal cations Ca²⁺, Mg²⁺ and transition metals Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ on DNA condensation and its protection against thermal denaturation in presence of dioleoylphosphatidylcholine liposomes (DOPC). Experimental results have shown that Ca²⁺ and Mg²⁺ as well as Zn²⁺ mediate DNA condensation. Cu²⁺ causes DNA double helix destabilization, and does not mediate binding between DNA and DOPC liposomes. Co²⁺ and Ni²⁺ can interact with DNA on both ways mentioned above. Static light scattering was use to follow the size of aggregates in DNA condensation process. Phospholipid bilayer and divalent cations protect condensed DNA against thermal destabilization. The highest stabilization effect was found in aggregates with Ca²⁺ and Zn²⁺, whereas in presence of either Co²⁺ or Ni²⁺ some volume fraction of DNA is denatured.

Stability of Human Growth Hormone: Influence of Methionine Oxidation on Thermal Folding

Oxidation, particularly of methionine residues, is one of the major chemical degradations of proteins. In a previous publication we studied the conformation of recombinant human growth hormone (r-hGH) selectively oxidized at Met14 and Met125. Conformation of oxidized r-hGH was found not different from that of nonoxidized r-hGH. In this paper, the effect of methionine oxidation on the thermal stability of r-hGH folding was investigated. The thermally induced unfolding process of the oxidized and nonoxidized protein was measured by monitoring the circular dichroism signal at 220 nm. The melting temperatures (T(m)) of the oxidized and nonoxidized r-hGH forms were determined at different pHs and in the presence of salts often used in pharmaceutical formulations. The effect of the location of the oxidized Met residue in the protein and the percentage of oxidation were investigated. Our findings indicate that the monoxidation of the two most accessible methionine residues of r-hGH-Met14 and Met125 - has no effect on the protein conformation. However, oxidation of these residues to form sulfoxides does influence the thermal stability of the protein folding. The presence of the polar oxygen atom on the methionine sulfoxide group thermally destabilizes r-hGH folding. The effect (T(m)) depends upon pH, ionic strength, and the location of the oxidized methionine residues in the protein. The thermal melting of r-hGH and its oxidized products is a highly cooperative process. Methionine oxidation leads to a thermal destabilization of the whole protein folding and is not just a local destabilization.

The effect of nanoparticles on structural morphology, thermal and flammability properties of two epoxy resins with different functionalities

The effect of layered silicate nanoclays, nano-silica and double-walled carbon nanotubes (DWNTs) on the thermal stability and fire reaction properties of two aerospace grade epoxy resins (a high temperature curing tetra-functional and a low temperature curing bi-functional resin) has been investigated using thermal analysis, cone calorimetry, LOI and UL-94 techniques. The morphology of the polymer–clay nanocomposites, determined by X-ray diffraction and transmission electron microscopy indicated intercalated structures. The addition of nanoclays (5-wt%) to both resins had a thermal destabilisation effect in the low temperature regime (<400 °C), but led to higher char yield at higher temperatures. The inclusion of nano-silica at 30-wt% significantly improved the thermal stability of the resins while DWNTs had an adverse effect due to their poor dispersion in the matrix. The nanoclays and carbon nanotubes significantly increased the fire resistance of the tetra-functional epoxy resin while a minimal effect was observed for the bi-functional resin.

The effect of GlycoPEGylation on the physical stability of human rFVIIa with increasing calcium chloride concentration

The effects of calcium chloride on the structural, kinetic and thermal stability of recombinant human factor VIIa (rFVIIa) were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10 kDa PEG and a branched 40 kDa PEG, respectively. Three different CaCl 2 concentrations were used: 10 mM, 35 mM and 100 mM. The secondary structure and tertiary structure of rFVIIa at 25 °C, measured by circular dichroism (CD), were maintained upon GlycoPEGylation as well as CaCl 2 content. In contrast, the thermal stability of the three rFVIIa compounds, measured by differential scanning calorimetry (DSC) and circular dichroism (CD), and aggregation behaviour, measured by light scattering (LS), were affected by the increasing calcium concentration. Increasing the CaCl 2 concentration from 10 mM to 35 mM resulted in a decrease in the apparent unfolding temperature, T m, of rFVIIa, whereas the concentration of CaCl 2 has to be raised to 100 mM in order to see the same effect on the GlycoPEGylated rFVIIa compounds. The temperature of aggregation of rFVIIa, T agg, increased as the CaCl 2 concentration increased from 35 mM to 100 mM, while T agg for the GlycoPEGylated rFVIIa compounds was practically independent of the CaCl 2 concentration. From the obtained results, it is concluded that GlycoPEGylation postpones the calcium induced thermal destabilisation of rFVIIa, and a much higher calcium concentration also postpones the thermally induced aggregation of rFVIIa. The thermally induced aggregation of the GlycoPEGylated rFVIIa compounds is unaffected by an increasing calcium chloride concentration.

Interactions of p-coumaric, caffeic and ferulic acids and their styrenes with model lipid membranes

The influence of the p-coumaric, caffeic and ferulic phenolic acids, and their corresponding styrenes, on structural properties of a model lipid membrane was investigated by differential scanning calorimetry and fluorimetry. The phenolic acids were seen to have smaller effects, compared to their styrenes, on the temperature and enthalpy of the main phase transition (gel-to-liquid) of extruded large unilamellar liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids. A slight thermal and enthalpy destabilisation of the liposomes was seen with the phenolic acids at a lipid:acid molar ratio of 1:1. In contrast, their styrenes had higher destabilisation effects on the enthalpy of the phase transition. For fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene as the membrane probe, the polarisation measurements showed that the styrenes, which are less polar than their corresponding acids, had greater effects on lipid membrane structure. The correlations between the structural properties of these compounds and their effects on membrane structure are discussed.