The consensus concept for thermostability engineering of proteins

Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (T(m)) of 78.0 degrees C which is 15-22 degrees C higher than the T(m) values of all parent phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus phytases with T(m) values of up to 90.4 degrees C. These increases in T(m) are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37 degrees C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins.     

Protein engineering for thermostability.

Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins.     

Glucose isomerase: insights into protein engineering for increased thermostability

Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.     

DNA watermarks: A proof of concept

Background  

DNA-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. In silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the DNA-Crypt algorithm.     

Physics-based gene identification: proof of concept for Plasmodium falciparum

The ab initio prediction of new genes in eukaryotic genomes represents a difficult task, notably for the identification of complex split genes. A Physics-Based Gene Identification (PBGI) method was formulated recently (Yeramian, Gene, 255, 139-150, 151-168, 2000a,b) to address this problem, taking as a model the Plasmodium falciparum genome. Here, the predictive power of this method is put under experimental test for this genome. The presented results demonstrate the usefulness of the PBGI as a gene-identification tool for P. falciparum, notably for the discovery of new genes with no homology to known genes. Perspectives opened by this new method for other eukaryotic genomes are also mentioned.     

Some thermodynamic implications for the thermostability of proteins

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* approximately 283K, independent of the heat denaturation temperature, T(m). This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of approximately 0.008 kJ/mole/residue is, on average, associated with a 1 degrees C increase in T(m). An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.     

Aerobic respiration: proof of concept for the oxygen-centric murburn perspective

Abstract

The inner mitochondrial membrane protein complexes (I–V) and prokaryotic respiratory machinery are examined for a deeper understanding of their structure–function correlations and dynamics. In silico analysis of the structure of complexes I–IV, docking studies and erstwhile literature confirm that they carry sites which are in close proximity to DROS (diffusible reactive oxygen species) generating redox centers. These findings provide supportive evidence for the newly proposed oxygen-centric chemical-coupling mechanism (murburn concept), wherein DROS catalyzes the esterification of inorganic phosphate to ADP. Further, in a reductionist system, we demonstrate that a DROS (like superoxide) can effectively esterify inorganic phosphate to ADP. The impact of these findings and the interactive dynamics of classical inhibitors (rotenone and cyanide), uncouplers (dinitrophenol and uncoupling protein) and other toxins (atractyloside and oligomycin) are briefly discussed.

Highlights

? Earlier perception: Complexes (I–IV) pump protons and Complex V make ATP (aided by protons)

? Herein: Respiratory molecular machinery is probed for new structure–function correlations

? Analyses: Quantitative arguments discount proton-centric ATP synthesis in mitochondria and bacteria

? In silico data: ADP-binding sites and O₂/ diffusible reactive oxygen species (DROS)-accessible channels are unveiled in respiratory proteins

? In vitro data: Using luminometry, ATP synthesis is demonstrated from ADP, Pi and superoxide

? Inference: Findings agree with decentralized ADP–Pi activation via oxygen-centric murburn scheme

Communicated by Ramaswamy H. Sarma     

The race concept in six regions: variation without consensus

Race, once the central concept in physical anthropology worldwide, now varies in the degree of support it receives in different regions. We present the currently available information on the status of the concept in the United States, the Spanish language areas, Poland, Europe, Russia, and China. Rejection of race ranges from high to low with the highest rejection occurring among anthropologists in the United States (and Canada). Rejection of race is moderate in Europe, sizeable in Poland and Cuba, and lowest in Russia and China. A discussion on the scientific and contextual reasons influencing these variations is presented. The tension between scientific evidence and social influences varies from region to region. The methods used in the studies reported here included questionnaires and content analysis. Response rates to questionnaires were often around 50 percent (with exception of the Polish studies). We discuss reasons for the low rates. Although a uniform method of data gathering is desirable, it may not suit scientists working in different traditions of theory and research. We conclude that it is once again timely to discuss the race concept in international meetings where all scientific and political changes occurring throughout the world in recent past decades are taken into account.     

Engineering proteins for thermostability through rigidifying flexible sites

Engineering proteins for thermostability is an exciting and challenging field since it is critical for broadening the industrial use of recombinant proteins. Thermostability of proteins arises from the simultaneous effect of several forces such as hydrophobic interactions, disulfide bonds, salt bridges and hydrogen bonds. All of these interactions lead to decreased flexibility of polypeptide chain. Structural studies of mesophilic and thermophilic proteins showed that the latter need more rigid structures to compensate for increased thermal fluctuations. Hence flexibility can be an indicator to pinpoint weak spots for enhancing thermostability of enzymes. A strategy has been proven effective in enhancing proteins' thermostability with two steps: predict flexible sites of proteins firstly and then rigidify these sites. We refer to this approach as rigidify flexible sites (RFS) and give an overview of such a method through summarizing the methods to predict flexibility of a protein, the methods to rigidify residues with high flexibility and successful cases regarding enhancing thermostability of proteins using RFS.     

The next 15 years: taking plant-made vaccines beyond proof of concept

Significant potential advantages are associated with the production of vaccines in transgenic plants; however, no commercial product has emerged. An analysis of the strengths, weaknesses, opportunities and threats for plant-made vaccine technology is provided. The use of this technology for human vaccines will require significant investment and developmental efforts that cannot be supported entirely by the academic sector and is not currently supported financially by industry. A focus on downstream aspects to define potential products, conduct of additional basic clinical testing, and the incorporation of multidisciplinary strategic planning would accelerate the potential for commercialization in this field. Estimates of production cost per dose and volume of production are highly variable for a model vaccine produced in transgenic tomato, and can be influenced by the optimization of many factors. Commercialization of plant-made vaccine technology is likely to be led by the agricultural biotechnology sector rather than the pharmaceutical sector due to the disruptive nature of the technology and the complex intellectual property landscape. The next major milestones will be conduct of a phase II human clinical trial and demonstration of protection in humans. The achievement of these milestones would be accelerated by further basic investigation into mucosal immunity, the codevelopment of oral adjuvants, and the integration of quality control standards and good manufacturing practices for the production of preclinical and clinical batch materials.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.     

Bioreactors for plant engineering: an outlook for further research

This paper reviews bioreactor related aspects of large scale plant cell technology for the production of biologically active compounds. Bioreactor designs currently in use are discussed with respect to specific operating parameters that can be varied to modulate cell growth and function in order to optimize product release and separation. Flow and mixing are recognized as key factors responsible for both the direct hydrodynamic effects on cell shape and function and flow induced changes in mass transfer of nutrients and metabolites. The integration of biosynthesis and separation is considered as a possible approach towards more efficient plant cell and tissue culture.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

Experiment-guided thermodynamic simulations on reversible two-state proteins: implications for protein thermostability

Here, we perform protein thermodynamic simulations within a set of boundary conditions, effectively blanketing the experimental data. The thermodynamic parameters, melting temperature (TG), enthalpy change at the melting temperature (DeltaHG) and heat capacity change (DeltaCp) were systematically varied over the experimentally observed ranges for small single domain reversible two-state proteins. Parameter sets that satisfy the Gibbs-Helmholtz equation and yield a temperature of maximal stability (TS) around room temperature were selected. The results were divided into three categories by arbitrarily chosen TG ranges. The TG ranges in these categories correspond to typical values of the melting temperatures observed for the majority of the proteins from mesophilic, thermophilic and hyperthermophilic organisms. As expected, DeltaCp values tend to be high in mesophiles and low in hyperthermophiles. An increase in TG is accompanied by an up-shift and broadening of the protein stability curves, however, with a large scatter. Furthermore, the simulations reveal that the average DeltaHG increases with TG up to approximately 360 K and becomes constant thereafter. DeltaCp decreases with TG with different rates before and after approximately 360 K. This provides further justification for the separate grouping of proteins into thermophiles and hyperthermophiles to assess their thermodynamic differences. This analysis of the Gibbs-Helmholtz equation has allowed us to study the interdependence of the thermodynamic parameters TG, DeltaHG and DeltaCp and their derivatives in a more rigorous way than possible by the limited experimental protein thermodynamics data available in the literature. The results provide new insights into protein thermostability and suggest potential strategies for its manipulation.     

How to reach linguistic consensus: a proof of convergence for the naming game

In this paper we introduce a mathematical model of naming games. Naming games have been widely used within research on the origins and evolution of language. Despite the many interesting empirical results these studies have produced, most of this research lacks a formal elucidating theory. In this paper we show how a population of agents can reach linguistic consensus, i.e. learn to use one common language to communicate with one another. Our approach differs from existing formal work in two important ways: one, we relax the too strong assumption that an agent samples infinitely often during each time interval. This assumption is usually made to guarantee convergence of an empirical learning process to a deterministic dynamical system. Two, we provide a proof that under these new realistic conditions, our model converges to a common language for the entire population of agents. Finally the model is experimentally validated.     

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.     

Tentative rules for increasing the thermostability of enzymes by protein engineering

The advent of recombinant DNA techniques provides protein chemistry with a powerful tool for designing and modifying, via site-directed mutagenesis, the physicochemical characteristics of enzymes. Among these characteristics is thermostability. Since site-directed mutagenesis has to be applied to replace as few amino acids as possible, it is necessary to know the rules that govern protein thermostability. To gain insight into these rules, we have performed the analysis of replacements between mesostable/thermostable counterparts of isoenzymes, based on a table of replacements for tyrosinases of Neurospora crassa. Upon prediction of the secondary structure and hydropathic profiles, we found that replacements are conservative in type and length of secondary structure and that they occur preferentially in external regions of the proteins. Some tentative rules for applying site-directed mutagenesis to proteins are proposed and discussed.