Protein thermostability above 100°C: A key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Background: HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. FINDINGS We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4[degree sign]C, --20[degree sign]C and -70[degree sign]C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70[degree sign]C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70[degree sign]C for 12 months is most effective in retaining VLP stability.

The difference of thermal stability between Fe-substituted palygorskite and Al-rich palygorskite

Sedimentary palygorskite (SP) and hydrothermal palygorskite (HP) were characterized by XRF, TG/DSC, andXRD. The total iron and dissociative iron in palygorskite were detected using spectrophotometry. The results showed that about 3.57 wt% of Fe2O3 was detected in SP in contrast with 0.4 wt% in HP. SP was a Fe-substituted palygorskite, and HP was an Al-rich palygorskite. The occurrence of Fe substitution in SP resulted in two mass loss steps of coordinated water and resulted in a larger d spacing. The SP showed greater thermal stability than the HP. It was proposed the change of (200) diffraction peak and (240) diffraction peak reflect changes of tetrahedral and octahedral structures in palygorskite.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of doublestranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 59 thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

Opsin clines in butterflies suggest novel roles for insect photopigments

Opsins are ancient molecules that enable animal vision by coupling to a vitamin-derived chromophore to form lightsensitive photopigments. The primary drivers of evolutionary diversification in opsins are thought to be visual tasks related to spectral sensitivity and color vision. Typically, only a few opsin amino acid sites affect photopigment spectral sensitivity. We show that opsin genes of the North American butterfly Limenitis arthemis have diversified along a latitudinal cline, consistent with natural selection due to environmental factors. We sequenced single nucleotide(SNP) polymorphisms in the coding regions of the ultraviolet (UVRh), blue (BRh), and long-wavelength (LWRh) opsin genes from ten butterfly populations along the eastern United States and found that a majority of opsin SNPs showed significant clinal variation. Outlier detection and analysis of molecular variance indicated that many SNPs are under balancing selection and show significant population structure. This contrasts with what we found by analysing SNPs in the wingless and EF-1 alpha loci, and from neutral amplified fragment length polymorphisms, which show no evidence of significant locus-specific or genome-wide structure among populations. Using a combination of functional genetic and physiological approaches, including expression in cell culture, transgenic Drosophila, UV-visible spectroscopy, and optophysiology, we show that key BRh opsin SNPs that vary clinally have almost no effect on spectral sensitivity. Our results suggest that opsin diversification in this butterfly is more consistent with natural selection unrelated to spectral tuning. Some of the clinally varying SNPs may instead play a role in regulating opsin gene expression levels or the thermostability of the opsin protein. Lastly, we discuss the possibility that insect opsins might have important, yet-to-be elucidated, adaptive functions in mediating animal responses to abiotic factors, such as temperature or photoperiod.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C. The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.

Synthesis and spectral studies of polyamide-phosphate esters from phosphine-oxide-containing diols with aryl phosphorodichloridates and their thermal and flammability behaviour

Polyamide-phosphate esters were synthesized by interfacial polycondensation of aryl phosphorodichloridates with the diols of phenoxaphosphine and phosphine oxide in the presence of a phase-transfer catalyst. The polymers were characterized by infra-red and 1H, 13C and 31P nuclear magnetic resonance (n.m.r.) spectroscopy. The molecular weights were determined by end-group analysis using 31P n.m.r. spectral data. The phenoxaphosphine-containing polymers showed superior thermostability and flame retardancy over the phosphine-oxide-containing polymers.

Thermal degradation studies of para-substituted poly(styrene peroxide)s

The thermal degradation of a series of para-substituted poly(styrene peroxide)s with electron-donating [CH3, C(CH3)(3)] and electron-attracting (Br) substitutents are investigated by thermogravimetric analysis (TGA). The results indicate that the Hammett relationship can describe quantitatively the trends in maximum rate of polymer decomposition (T-max) observed in TGA and thus thermostability of substituted poly(styrene peroxide)s depends only on the electronic nature of substituents and their ability to stabilize macroradicals formed during chain scission. The experimental results are also substantiated by thermochemical calculations. (C) 2002 Elsevier Science Ltd. All rights reserved.

Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop.

Molecular Dynamics Perspective on the Protein Thermal Stability: A Case Study Using SAICAR Synthetase

The enzyme SAICAR synthetase ligates aspartate with CAIR (5'-phosphoribosyl-4-carboxy-5-aminoimidazole) forming SAICAR (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide) in the presence of ATP. In continuation with our previous study on the thermostability of this enzyme in hyper-/thermophiles based on the structural aspects, here, we present the dynamic aspects that differentiate the mesophilic (E. coli, E. chaffeensis), thermophilic (G. kaustophilus), and hyperthermophilic (M. jannaschii, P. horikoshii) SAICAR synthetases by carrying out a total of 11 simulations. The five functional dimers from the above organisms were simulated using molecular dynamics for a period of 50 ns each at 300 K, 363 K, and an additional simulation at 333 K for the thermophilic protein. The basic features like root-mean-square deviations, root-mean-square fluctuations, surface accessibility, and radius of gyration revealed the instability of mesophiles at 363 K. Mean square displacements establish the reduced flexibility of hyper-/thermophiles at all temperatures. At the simulations time scale considered here, the long-distance networks are considerably affected in mesophilic structures at 363 K. In mesophiles, a comparatively higher number of short-lived (having less percent existence time) C alpha, hydrogen bonds, hydrophobic interactions are formed, and long-lived (with higher percentage existence time) contacts are lost. The number of time-averaged salt-bridges is at least 2-fold higher in hyperthermophiles at 363 K. The change in surface accessibility of salt-bridges at 363 K from 300 K is nearly doubled in mesophilic protein compared to proteins from other temperature classes.