Thermal Destabilization Research Articles

(N7)-Platination and its effect on (N1)H-acidification in nucleoside phosphate derivatives

Pt(II) coordination, like of cis-(NH3)2Pt(II), affects the acid-base properties of guanosine derivatives. The acidity constants of such complexes are calculated by curve-fitting procedures using published 1H NMR shift data measured in aqueous solution (D2O) in dependence on pH (pD). Comparison of the pKa values of the ligands with those of the Pt(II) complexes reveals the expected behavior for (N7)-platination, i.e., (N1)H sites are acidified due to charge repulsion. These effects of Pt(II) are compared with those of Ni2+ and Cd2+, allowing predictions for the acidification of Zn2+. Studied are the cis-(NH3)2Pt(II) complexes of inosylyl(3′→5′)inosine, guanylyl(3′→5′)guanine (GpG)−, its 2′-deoxy relative d(GpG)−, and its phosphorylated derivative d(pGpG)3−. The Pt(II) effect is mostly rather symmetrical, like with d(CpGpG)2−, but it can also be quite asymmetrical, like with d(CpCpGpG)3− or in the cyclohexylamine-substituted Cisplatin complex of d(GpG)−. Potential consequences for base pairing between the platinated guanine and the complementary cytosine are discussed. It is proposed that partial deprotonation of dG under the influence of Pt(II) at physiological pH leads in water to a marked reduction in pairs with intact Watson–Crick hydrogen bonds, thereby adding to the well established thermal destabilization of double-stranded DNA by G,G intrastrand cross-links of Cisplatin.

Stabilization of the thermolabile variant S113L of carnitine palmitoyltransferase II.

Objective:Muscle carnitine palmitoyltransferase (CPT) II deficiency, the most common defect of lipid metabolism in muscle, is characterized by attacks of myoglobinuria without persistent muscle weakness.Methods:His6-N-hCPT2 (wild-type) and His6-N-hCPT2/S113L (variant) were produced recombinantly in prokaryotic host and characterized according to their functional and regulatory properties.Results:The wild-type and the variant S113L showed the same enzymatic activity and thermostability at 30°C. The mutated enzyme, however, revealed an abnormal thermal destabilization at 40°C and 45°C. This was consistent with an increased flexibility (B-factor) of the variant at 40°C compared with that of the wild-type shown by molecular dynamics analysis. Preincubation of the enzymes with l-carnitine and acyl-l-carnitines containing more than 10 carbons in the acyl side-chain stabilized the mutated enzyme against thermal inactivation. In contrast, palmitoyl-CoA destabilized both enzymes.Conclusions:The problems in CPT II deficiency originating from the S113L mutation are not caused by the loss of catalytically active enzyme. They might be at least partially related to an impaired thermal stability of the protein. The lower thermodynamic stability of the variant might explain why fever and prolonged exertion provoke attacks of myoglobinuria in CPT II deficiency. The stabilization by acyl-l-carnitines might provide the basis for possible preventive therapy of CPT II deficiency.

Study of the effect of tannins and animal species on the thermal stability of vegetable leather by differential scanning calorimetry

Micro-differential scanning calorimetry was used to reveal the deterioration patterns of collagen in vegetable-tanned leather. The influence of both the tannin type, i.e. hydrolysable or condensed, and collagen animal species, i.e. calf and sheep, was investigated. Comparison with the behaviour of unmodified collagen in parchment was made to explain the thermal destabilisation and denaturation of the chemically modified collagen in leather. Both leather and parchment were subjected to accelerate ageing by heating at 70 °C in controlled atmosphere at 30 % RH. The synergistic effect of the daylight exposure was studied by irradiating the samples in the visible domain with 4000 lx. The destabilisation effect induced by the hydrothermal ageing treatment was evident since the 8th day and reached a critical level after 32-day ageing time. The formation of damaged intermediate states with progressively lower thermal stability was the main feature of the deterioration pattern independent of the tannin type and collagen species. Quebracho-tanned calf leather was the most resistant against ageing, whereas chestnut-tanned sheep leather underwent de-tanning after a 32-day ageing period. Exposure to visible light irradiation induced an evident thermal stabilisation due to cross-link formation. The balance between thermal stabilisation and destabilisation processes in leather during visible light exposure was influenced by the tannin type.

Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw.

This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed.

Amphibole reaction rims as a record of pre-eruptive magmatic heating: An experimental approach

Magmatic minerals record the pre-eruptive timescales of magma ascent and mixing in crustal reservoirs and conduits. Investigations of the mineral records of magmatic processes are fundamental to our understanding of what controls eruption style, as ascent rates and magma mixing processes are well known to control and/or trigger potentially hazardous explosive eruptions. Thus, amphibole reaction rims are often used to infer pre-eruptive magma dynamics, and in particular to estimate magma ascent rates. However, while several experimental studies have investigated amphibole destabilization during decompression, only two investigated thermal destabilization relevant to magma mixing processes. This study examines amphibole decomposition experimentally through isobaric heating of magnesio-hornblende phenocrysts within a natural high-silica andesite glass. The experiments first equilibrated for 24 h at 870 °C and 140 MPa at H2O-saturated conditions and ƒO2 ∼ Re–ReO prior to rapid heating to 880, 900, or 920 °C and hold times of 3–48 h. At 920 °C, rim thicknesses increased from 17 μm after 3 h, to 55 μm after 12 h, and became pseudomorphs after longer durations. At 900 °C, rim thicknesses increased from 7 μm after 3 h, to 80 μm after 24 h, to pseudomorphs after longer durations. At 880 °C, rim thicknesses increased from 7 μm after 3 h, to 18 μm after 36 h, to pseudomorphs after 48 h. Reaction rim microlites vary from 5–16 μm in size, with no systematic relationship between crystal size and the duration or magnitude of heating. Time-averaged rim microlite growth rates decrease steadily with increasing experimental duration (from 3.97×10−7 mms−1 to 3.1 to 3.5×10−8 mms−1). Time-averaged microlite nucleation rates also decrease with increasing experimental duration (from 1.2×103 mm−3s−1 to 5.3 mm−3 s−1). There is no systematic relationship between time-averaged growth or nucleation rates and the magnitude of the heating step. Ortho- and clinopyroxene together constitute 57–90 modal % mineralogy in each reaction rim. At constant temperature, clinopyroxene abundances decrease with increasing experimental duration, from 72 modal % (3 h at 900 °C) to 0% (48 h at 880 °C, and 36 h at 900 and 920 °C). Fe–Ti oxides increase from 6–12 modal % (after 3–6 h) to 26–34 modal % (after 36–48 h). Plagioclase occurs in relatively minor amounts (<1–11 modal %), with anorthite contents that increase from An56 to An88 from 3 to 36 h of heating. Distal glass compositions (>500 μm from reacted amphibole) are consistent with inter-microlite rim glasses (71.3–77.7 wt.% SiO2) within a given experiment and there is a weakly positive correlation between increasing run duration and inter-microlite melt SiO2 (68.9–78.5 wt.%). Our results indicate that experimental heating-induced amphibole reaction rims have thicknesses, textures, and mineralogies consistent with many of the natural reaction rims seen at arc-andesite volcanoes. They are also texturally consistent with experimental decompression reaction rims. On this basis it may be challenging to distinguish between decompression and heating mechanisms in nature.

Tuning the pH Response of i-Motif DNA Oligonucleotides.

Cytosine-rich single-stranded DNA oligonucleotides are able to adopt an i-motif conformation, a four-stranded structure, near a pH of 6. This unique pH-dependent conformational switch is reversible and hence can be controlled by changing the pH. Here, we show that the pH response range of the human telomeric i-motif can be shifted towards more basic pH values by introducing 5-methylcytidines (5-MeC) and towards more acidic pH values by introducing 5-bromocytidines (5-BrC). No thermal destabilisation was observed in these chemically modified i-motif sequences. The time required to attain the new conformation in response to sudden pH changes was slow for all investigated sequences but was found to be ten times faster in the 5-BrC derivative of the i-motif.

Impact of glycosylation on stability, structure and unfolding of soybean agglutinin (SBA): an insight from thermal perturbation molecular dynamics simulations.

Glycosylation has been recognized as one of the most prevalent and complex post-translational modifications of proteins involving numerous enzymes and substrates. Its effect on the protein conformational transitions is not clearly understood yet. In this study, we have examined the effect of glycosylation on protein stability using molecular dynamics simulation of legume lectin soybean agglutinin (SBA). Its glycosylated moiety consists of high mannose type N-linked glycan (Man9GlcNAc2). To unveil the structural perturbations during thermal unfolding of these two forms, we have studied and compared them to the experimental results. From the perspective of dynamics, our simulations revealed that the nonglycosylated monomeric form is less stable than corresponding glycosylated form at normal and elevated temperatures. Moreover, at elevated temperature thermal destabilization is more prominent in solvent exposed loops, turns and ends of distinct β sheets. SBA maintains it folded structure due to some important saltbridges, hydrogen bonds and hydrophobic interactions within the protein. The reducing terminal GlcNAc residues interact with the protein residues VAL161, PRO182 and SER225 via hydrophobic and via hydrogen bonding with ASN 9 and ASN 75. Our simulations also revealed that single glycosylation (ASN75) has no significant effect on corresponding cis peptide angle orientation. This atomistic description might have important implications for understanding the functionality and stability of Soybean agglutinin.

Kaempferol and quercetin interactions with model lipid membranes

In the present study, the membrane interactions of the flavonoids kaempferol and quercetin at different temperatures with two different model lipid membranes (without cholesterol) were investigated. A combination of fluorescence spectroscopy, electron paramagnetic resonance spectroscopy, and differential scanning calorimetry were used to detect changes in membrane fluidity. Two fluorophores and two spin probes were used to monitor the membrane characteristics at different depths in the membrane. The dependence of the lipid order on the molar ratio of flavonoid to membranes of phosphatidylcholine:sphingomyelin (2.4:1) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine showed that flavonoids increase the lipid order. As with fluorescence spectroscopy, for phosphatidylcholine:sphingomyelin liposomes, the fluidity decrease detected with the spin probes of the electron paramagnetic resonance spectroscopy was less with kaempferol than quercetin, and was higher in the middle of the membrane than close to the water–lipid interface. Below the phase-transition temperature, the polarity surrounding the nitroxide group of the spin probes in the most-ordered membrane domains increases in the presence of kaempferol and quercetin, which indicates that these flavonoids facilitate the penetration of water molecules into the membranes. Kaempferol and quercetin caused a small thermal destabilisation of DPPC transition which shows that the molecules interacted mainly with the polar headgroup regions of the bilayer. With the kaempferol and quercetin, which differ only in their number of –OH groups, their effect on the lipid membranes was more pronounced in the absence of cholesterol and confirmed that even a difference of one –OH group is enough to promote different membrane effects.

Engineering Giardia lamblia trimethylguanosine synthase (GlaTgs2) to transfer non-natural modifications to the RNA 5'-cap.

Trimethylguanosine synthase from Giardia lamblia (GlaTgs2) naturally catalyzes methyl transfer from S-adenosyl-L-methionine (AdoMet) to the exocyclic N(2) atom of the 5'-cap--a hallmark of eukaryotic mRNAs. The wild-type enzyme shows substrate promiscuity and can also use the AdoMet-analog AdoPropen for allyl transfer. Here we report on engineering GlaTgs2 to enhance the activity on AdoPropen. A mutational analysis, involving an alanine scan of 10 residues located around the active site, was performed. Positions V34 and S38 were identified as mutational hot spots and analyzed in greater detail by testing NNK libraries. Kinetic analysis and thermostability measurements revealed V34A as the best variant of GlaTgs2, with a ∼10-fold improved specificity for AdoPropen. Double mutants did not yield additional improvements due to low catalytic efficiencies and thermal destabilization. Homologous Tgs enzymes from Homo sapiens and G. intestinalis were also investigated regarding their catalytic activity on AdoPropen. While neither the human wild-type (WT) enzyme nor any of its variants showed activity on AdoPropen, the homologue from G. intestinalis (GinTgs) was remarkably active on AdoPropen. Introducing the best substitution at the homologous position led to variant T34A with ∼40-fold higher specificity for AdoPropen than the original GlaTgs2 WT.

Thermal Stability of the Commercial Mg‐PSZ Powders

Using X‐ray diffraction and scanning electron microscopy two commercial Mg‐PSZ ceramics were studied regarding their thermal stability at 1350 °C. The Mg‐PSZ material was investigated in as‐received, sintered, and powdered states. Drastic increasing of the m‐ZrO2 content was observed for the as‐received powders. Whereas the Mg‐PSZ ceramics sintered at 1600 °C exhibit certainly better thermal stability. Two mechanisms of thermal destabilization were defined using observations of the sintered Mg‐PSZ material. Influence of the particle size of Mg‐PSZ on its thermal stability is defined.

Low pH Modulates the Macroorganization and Thermal Stability of PSII Supercomplexes in Grana Membranes

Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes—formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching.

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.

Functionalization of graphene with grafted polyphosphamide for flame retardant epoxy composites: synthesis, flammability and mechanism

A polyphosphamide (PPA) was synthesized and covalently grafted onto the surface of graphene nanosheets (GNSs) to obtain a novel flame retardant, PPA-g-GNS, and subsequently PPA-g-GNS was incorporated into epoxy resins (EPs) to enhance the fire resistance. The chemical structures and morphology of the precursors and target product were confirmed using 1H-NMR spectroscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The tensile results showed that the mechanical strength and modulus of the PPA-g-GNS/EP composite were higher than those of pure EP and PPA/EP, owing to the outstanding reinforced effect of graphene. The evaluation of the thermal properties demonstrated that the addition of PPA or PPA-g-GNS to epoxy had a thermal destabilization effect below 400 °C, but led to a higher char yield at higher temperatures. Furthermore, the PPA-g-GNS/EP composite exhibited superior fire resistant performance, such as higher LOI values and reduced PHRR and FIGRA values, compared to pure EP and PPA/EP, which was probably attributed to the higher char yield during combustion. A possible flame retardant mechanism was speculated according to the direct pyrolysis-mass spectrometry results: the phosphate species degraded from PPA catalyzed the decomposition of the PPA-g-GNS/EP composites to generate various pyrolysis products; the pyrolysis products were absorbed and propagated on the graphene which served as a template of micro-char, and thus a continuous and compact char layer was formed; such a char layer provided effective shields to protect the underlying polymers against flame.

Thickness-dependent effects in H2O desorption from glycine/amorphous solid water films

The desorption characteristics of H2O from glycine-on-amorphous solid water (ASW) ultrathin films have been systematically investigated as a function of underlying ASW thickness and the amount of top-deposited glycine using TPD. At the early stages of deposition, substantial amounts of glycine molecules penetrate into the water multilayers and, therefore, thermal destabilization of water layers is detected. Glycine diffusion is governed by the thickness-dependent structure of the ASW as revealed by work function measurements and XPS. For higher glycine coverages, a kinetic restriction of water desorption by the continuous amino acid overlayers is observed independent of the ASW substrate thickness.

The effects of rapid solidification on microstructure and hydrogen sorption properties of binary BCC Ti–V alloys

Abstract The main purpose of the present work was to study the effect of rapid solidification (RS) on the microstructure and hydrogen storage properties of body centred cubic (BCC) Ti rich Ti–V alloys (Ti1−xVx, x = 0.1–0.3). Ribbons were prepared by melt spinning at spinner rotation velocities of 1000–3000 rpm. Ribbon morphology and microstructure were found to depend on the vanadium content and spinner velocity. For Ti0.8V0.2, the relation between the ribbon thickness and velocity can be expressed as a power law function, indicating that, during solidification of the Ti–V ribbons, heat transfer at the interface between spinner and ribbon controls the heat extraction. Temperature desorption spectroscopy (TDS) and in situ synchrotron (SR-XRD) studies of the RS alloys showed that hydrogen desorption from the RS alloy hydrides occurred at lower temperatures than from the as cast alloys. RS caused a microscale chemical element separation in the alloys, which depends on the vanadium content and the spinner velocity. In addition, ribbon recalescence was observed to cause nanoscale chemical redistribution trough spinodal decomposition. These two last features were proposed to be the reasons for the observed thermal destabilisation.

Cross‐link stabilization does not affect the response of collagen molecules, fibrils, or tendons to tensile overload

We investigated whether immature allysine-derived cross-links provide mechanically labile linkages by exploring the effects of immature cross-link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH₄) to stabilize labile cross-links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One-half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH4 cross-link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross-link stabilization did not prevent the characteristic overload-induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload-induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross-links may not be as mechanically labile as was previously thought.

Structural and Thermodynamic Insight into Escherichia coli UvrABC-Mediated Incision of Cluster Diacetylaminofluorene Adducts on the NarI Sequence

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

The effect of graphene presence in flame retarded epoxy resin matrix on the mechanical and flammability properties of glass fiber-reinforced composites

The effect of adding graphene in epoxy containing either an additive (MP) or reactive-type (DOPO) flame retardant on the thermal, mechanical and flammability properties of glass fiber-reinforced epoxy composites was investigated using thermal analysis; flexural, impact, tensile tests; cone calorimetry and UL-94 techniques. The addition of MP or DOPO to epoxy had a thermal destabilization effect below 400°C, but led to higher char yield at higher temperatures. The inclusion of 10wt% flame retardants slightly decreased the mechanical behavior, which was attributed to the poor interfacial interactions in case of MP or the decreased cross-linking density in case of DOPO flame retarded resin. The additional graphene presence increased flexural and impact properties, but slightly decreased tensile performance. Adding graphene further decreased the PHRR, THR and burning rate due to its good barrier effect. The improved fire retardancy was mainly attributed to the reduced release of the combustible gas products.

Comparison of Cis‐ and Oxaliplatin‐induced Destabilization of 15‐mer DNA‐ and RNA Duplexes by Binding to Centrally Located GG‐ and GNG Sequences

AbstractThermodynamic parameters are presented here illustrating the effects caused by the two anticancer active metal complexes cisplatin and oxaliplatin after introduction into four closely related RNA and DNA duplexes. The duplexes used are blunt end, fully complementary 15‐mer duplexes with a centrally located either GG‐ or GNG (here: N = T or U) binding site. For all duplexes, a common trend of reduced melting temperature was observed after platination. Analysis of the thermodynamic parameters for the duplex dissociation reactions showed good correlation between variations in melting temperatures (Tm) and ground state enthalpies (ΔH) in both DNA‐ and RNA duplexes. The melting temperatures of the native duplexes were found to be determined by their chemical nature, i.e. with observed Tm ‐values of ca. 50 °C for DNA and ca. 61 °C for RNA (CT = 2 μM and CNa+ = 129 mM, pH 6.3). Of the two types of nucleic acids, RNA is the one that exhibits the most pronounced sensitivity towards introduction of the platinum complexes, and with oxaliplatin as the more influential metalation reagent. Of note is that the thermal destabilization caused by oxaliplatin interacting with a centrally located GUG‐sequence results in a duplex stability below native DNA.

An unusual effect of NADP+ on the thermostability of the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Adiabatic differential scanning calorimetry was used to investigate the effect of NADP+ on the irreversible thermal denaturation of the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans. The GAPN-NADP+ binary complex showed a strongly decreased thermal stability, with a difference of about 20 °C between the temperatures of the thermal transition peak maxima of the complex and the free protein. This finding was similar to the previously described thermal destabilization of GAPN upon binding of inorganic phosphate to the substrate binding site and can be interpreted as the shift of the equilibrium between 2 conformers of tetrameric GAPN upon addition of the coenzyme. Single amino acid substitution, known to abolish the NADP+ binding, cancelled the calorimetric effect of the coenzyme. GAPN thermal inactivation was considerably decelerated in the presence of NADP+ showing that the apparent change in stability of the active centre can be the opposite to that of the whole protein molecule. NADP+ could also reactivate the inactive GAPN* species, obtained by the heating of the apoenzyme below the thermal denaturation transition temperature. These effects may reflect a mechanism that provides GAPN the sufficient flexibility for the earlier observed profound active site reorganizations required during the catalytic cycle. The elevated thermal stability of the apoenzyme may, in turn, be important for maintaining a constant level of active GAPN--an enzyme that is known to be crucial for the effective supply of the reducing equivalents in S. mutans and its ability to grow under aerobic conditions.