Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands β₅ and β₆, loop L₅ and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.     

Protein thermal stability: insights from atomic displacement parameters (B values)

The factors contributing to the thermal stability of proteins from thermophilic origins are matters of intense debate and investigation. Thermophilic proteins are thought to possess better packed interiors than their mesophilic counterparts, leading to lesser overall flexibility and a corresponding reduction in surface-to-volume ratio. These observations prompted an analysis of B values reported in high-resolution X-ray crystal structures of mesophilic and thermophilic proteins. In this analysis, the following aspects were addressed: (1) frequency distribution of normalized B values (B' factors) over all the proteins and for individual amino acids; (2) amino acid compositions in high B value regions of polypeptide chains; (3) variation in the B values from core to the surface of proteins in terms of their radius of gyration; and (4) degree of dispersion of normalized B values in spheres around the Calpha atoms. The analysis revealed that (1) Ser and Thr have lesser flexibility in thermophiles than in mesophiles, (2) the proportion of Glu and Lys in high B value regions of thermophiles is higher and that of Ser and Thr is lower and (3) the dispersion of B values within spheres at Calpha atoms is similar in mesophiles and thermophiles. These observations reflect plausible differences in the dynamics of thermophilic and mesophilic proteins and suggest amino acid substitutions that are likely to change thermal stability.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase

By comparing a mesophilic alpha-amylase with its thermophilic homolog, we investigated the relationship between thermal stability and internal equilibrium fluctuations. Fourier transform infrared spectroscopy monitoring hydrogen/deuterium (H/D) exchange kinetics and incoherent neutron scattering measuring picosecond dynamics were used to study dynamic features of the folded state at room temperature. Fairly similar rates of slowly exchanging amide protons indicate about the same free energy of stabilization DeltaG(stab) for both enzymes at room temperature. With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected higher structural flexibility as compared to the mesophilic counterpart. In particular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase. The mechanism proposed for increasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of DeltaG(stab) as a function of temperature.     

Finding the generalized molecular principles of protein thermal stability

Are there any generalized molecular principles of thermal adaptation? Here, integrating the concepts of structural bioinformatics, sequence analysis, and classical knot theory, we develop a robust computational framework that seeks for mechanisms of thermal adaptation by comparing orthologous mesophilic-thermophilic and mesophilic-hyperthermophilic proteins of remarkable structural and topological similarities, and still leads us to context-independent results. A comprehensive analysis of 4741 high-resolution, non-redundant X-ray crystallographic structures collected from 11 hyperthermophilic, 32 thermophilic and 53 mesophilic prokaryotes unravels at least five “nearly universal” signatures of thermal adaptation, irrespective of the enormous sequence, structure, and functional diversity of the proteins compared. A careful investigation further extracts a set of amino acid changes that can potentially enhance protein thermal stability, and remarkably, these mutations are overrepresented in protein crystallization experiments, in disorder-to-order transitions and in engineered thermostable variants of existing mesophilic proteins. These results could be helpful to find a precise, global picture of thermal adaptation.     

Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro: activation of an anti-inflammatory cyclooxygenase-2 (COX-2) pathway

Introduction  

The mono- and digalactosyldiacylglycerol (MGDG and DGDG) galactolipids have been purified from the thermophilic blue-green alga Phormidium sp. ETS-05 that colonizes the therapeutic thermal mud of Abano Terme and Montegrotto Terme, Italy. Both compounds present a marked composition in polyunsaturated fatty acids, mainly omega-3. The therapeutic thermal mud is applied mainly to osteoarthritic cartilage patients. In the present study the effect of MGDG treatment on proteins and factors expressed by human articular cartilage cells in culture and on pathways activated in inflammatory conditions was studied.     

Amino acid coupling patterns in thermophilic proteins

Structural analysis is useful in elucidating structural features responsible for enhanced thermal stability of proteins. However, due to the rapid increase of sequenced genomic data, there are far more protein sequences than the corresponding three-dimensional (3D) structures. The usual sequence-based amino acid composition analysis provides useful but simplified clues about the amino acid types related to thermal stability of proteins. In this work, we developed a statistical approach to identify the significant amino acid coupling sequence patterns in thermophilic proteins. The amino acid coupling sequence pattern is defined as any 2 types of amino acids separated by 1 or more amino acids. Using this approach, we construct the rho profiles for the coupling patterns. The rho value gives a measure of the relative occurrence of a coupling pattern in thermophiles compared with mesophiles. We found that thermophiles and mesophiles exhibit significant bias in their amino acid coupling patterns. We showed that such bias is mainly due to temperature adaptation instead of species or GC content variations. Though no single outstanding coupling pattern can adequately account for protein thermostability, we can use a group of amino acid coupling patterns having strong statistical significance (p values < 10(-7)) to distinguish between thermophilic and mesophilic proteins. We found a good correlation between the optimal growth temperatures of the genomes and the occurrences of the coupling patterns (the correlation coefficient is 0.89). Furthermore, we can separate the thermophilic proteins from their mesophilic orthologs using the amino acid coupling patterns. These results may be useful in the study of the enhanced stability of proteins from thermophiles-especially when structural information is scarce. Proteins 2005. (c) 2005 Wiley-Liss, Inc.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Robustness of predictions of extremely thermally stable proteins in ancient organisms

A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.     

Aromatic clusters: a determinant of thermal stability of thermophilic proteins

A number of factors have been elucidated as responsible for the thermal stability of thermophilic proteins. However, the contribution of aromatic interactions to thermal stability has not been systematically studied. In the present investigation we used a graph spectral method to identify aromatic clusters in a dataset of 24 protein families for which the crystal structures of both the thermophilic and their mesophilic homologues are known. Our analysis shows a presence of additional aromatic clusters or enlarged aromatic networks in 17 different thermophilic protein families, which are absent in the corresponding mesophilic homologue. The additional aromatic clusters identified in the thermophiles are smaller in size and are largely found on the protein surface. The aromatic clusters are found to be relatively rigid regions of the surface and often the additional aromatic cluster is located close to the active site of the thermophilic enzyme. The residues in the additional aromatic clusters are preferably mutated to Leu, Ser or Ile in the mesophilic homologue. An analysis of the packing geometry of the pairwise aromatic interaction in the additional aromatic clusters shows a preference for a T-shaped orthogonal packing geometry. The present study also provides new insights for protein engineers to design thermostable and thermophilic proteins.     

Steepness of thermal gradient is essential to obtain a unified view of thermotaxis in C. elegans

One of the adaptive behaviors of animals in their environment is thermotaxis, by which they migrate toward a preferred temperature. This sensorimotor integration is accomplished by choosing one of two behaviors depending on the surrounding temperature, namely thermophilic or cryophilic movement. Caenorhabditis elegans exhibits thermotaxis and its migration behavior has been analyzed experimentally at both the population and individual levels. However, some experimental data are inconsistent especially for thermophilic movement, which is expected to be observed in lower than favorable temperatures. There are no experimental analyzes that find thermophilic tendencies in the individual behavior of worms, despite multiple reports supporting thermophilic movement of the population. Although theoretical methods have been used to study thermotaxis of C. elegans, no mathematical model provides a consistent explanation for this discrepancy. Here we develop a simple biased random walk model, which describes population behavior, but which is based on the results of individual assays. Our model can integrate all previous experiments without any contradiction. We regenerate all the population patterns reported in past studies and give a consistent explanation for the conflicting results. Our results suggest that thermophilic movement is observed, even in individual movements, when the thermal gradient is sufficiently slight. On the contrary, thermophilic movement disappears when the thermal gradient is too steep. The thermal gradient is thus essential for a comprehensive understanding of the experimental studies of thermotaxis in C. elegans. Our model provides insight into an integrative understanding of the neural activity and thermotactic behavior in C. elegans.     

Atomic mean-square displacements in proteins by molecular dynamics: a case for analysis of variance

Information on protein internal motions is usually obtained through the analysis of atomic mean-square displacements, which are a measure of variability of the atomic positions distribution functions. We report a statistical approach to analyze molecular dynamics data on these displacements that is based on probability distribution functions. Using a technique inspired by the analysis of variance, we compute unbiased, reliable mean-square displacements of the atoms and analyze them statistically. We applied this procedure to characterize protein thermostability by comparing the results for a thermophilic enzyme and a mesophilic homolog. In agreement with previous experimental observations, our analysis suggests that the proteins surface regions can play a role in the different thermal behavior.     

Role of cation-pi interactions to the stability of thermophilic proteins

Elucidating the factors responsible for exhibiting extreme thermal stability of thermophilic proteins is very important for an understanding of the mechanism of protein stability, as well as to design stable proteins. In this work, we have analyzed the influence of cation-pi interactions to enhance the stability from mesophilic to thermophilic proteins. The favorable residue pairs forming such a system of interactions have been brought out. We found that the Tyr has a greater number of such interactions with Lys in thermophilic proteins. Specifically, the same Lys would experience a greater number of cation-pi interactions with several Tyr residues in thermophiles. On the other hand, the influence of Phe in making cation-pi interactions is higher in mesophiles than in thermophiles. Further, a network of cation-pi interactions are maintained by Lys in thermophiles, whereas Arg plays a major role in mesophilic proteins. Moreover, atoms that have a substantial positive charge in both Lys and Arg make a more significant contribution for cation-pi interactions than do cationic group atoms.     

Climate change reduces offspring fitness in littoral spawners: a study integrating organismic response and long‐term time‐series

Integrating long‐term ecological observations with experimental findings on species response and tolerance to environmental stress supports an understanding of climate effects on population dynamics. Here, we combine the two approaches, laboratory experiments and analysis of multi‐decadal time‐series, to understand the consequences of climate anomalies and ongoing change for the population dynamics of a eurythermal littoral species, Carcinus aestuarii. For the generation of cause and effect hypotheses we investigated the thermal response of crab embryos at four developmental stages. We first measured metabolic rate variations in embryos following acute warming (16–24 °C) and after incubation at 20 and 24 °C for limited periods. All experiments consistently revealed differential thermal responses depending on the developmental stage. Temperature‐induced changes in metabolic activity of early embryonic stages of blastula and gastrula suggested the onset of abnormal development. In contrast, later developmental stages, characterized by tissue and organ differentiation, were marginally affected by temperature anomalies, indicating enhanced resilience to thermal stress. Then, we extended these findings to a larger, population scale, by analyzing a time‐series of C. aestuarii landings in the Venice lagoon from 1945 to 2010 (ripe crabs were recorded separately) in relation to temperature. Landings and extreme climatic events showed marked long‐term and short‐term variations. We found negative relationships between landings and thermal stress indices on both timescales, with time lags consistent with an impact on crab early life stages. When quantitatively evaluating the influence of thermal stress on population dynamics, we found that it has a comparable effect to that of the biomass of spawners. This work provides strong evidence that physiological responses to climatic anomalies translate into population‐level changes and that apparently tolerant species may be impacted before the ontogeny of eurythermy. These ontogenetic bottlenecks markedly shape population dynamics and require study to assess the effects of global change.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T opt = 85 degrees C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.     

Explanation of the stability of thermophilic proteins based on unique micromorphology

Two mesophilic/thermophilic variants of the G-domain of the elongation factor Tu were studied via molecular dynamics simulations. By analyzing the simulation data via the Voronoi space tessellation, we have found that the two proteins have the same macromolecular packing, while the water-exposed surface area is larger for the thermophile. A larger coordination with water is probably due to a peculiar corrugation of the exposed surface of this species. From an enthalpic point of view, the thermophile shows a larger number of intramolecular hydrogen bonds, stronger electrostatic interactions, and a flatter free-energy landscape. Overall, the data suggest that the specific hydration state enhances macromolecular fluctuations but, at the same time, increases thermal stability.