Positive aspects of negative design: simultaneous selection of specificity and interaction stability

The energetic determinants that drive specific protein-protein interactions are not entirely understood. We describe simultaneous in vivo selection of specific and stable interactions using homologous peptides which compete with protein libraries for an interaction with a target molecule. Library members binding to their target, and promoting cell growth, must outcompete competitor interactions with the target (i.e., competition) and evade binding to the competitors (i.e., negative design). We term this a competitive and negative design initiative (CANDI). We combined CANDI with a protein-fragment complementation assay (PCA) and observed major specificity improvements, by driving selection of winning library members that bind their target with maximum efficacy, ensuring that otherwise energetically accessible alternatives are inaccessible. CANDI-PCA has been used with libraries targeted at coiled coil regions of oncogenic AP-1 components cJun and cFos. We demonstrate that comparable hydrophobic and electrostatic contributions in desired species are compromised in nondesired species when CANDI is executed, demonstrating that both core and electrostatic residues are required to direct specific interactions. Major energetic differences (>or=5.6 kcal/mol) are observed between desired and nondesired interaction stabilities for a CANDI-PCA derived peptide relative to a conventional PCA derived helix, with significantly more stability (3.2 kcal/mol) than the wild-type cJun-cFos complex. As a negative control, a library lacking a residue repertoire able to generate a specific and stable helix was tested. Negative protein design has broad implications in generating specific and therapeutically relevant peptide-based drugs, proteins able to act with minimal cross-talk to homologues or analogues, and in nanobiotechnological design.     

Arginyl residues and thermal stability in proteins

Guanidination and amidination of bovine serum albumin, yeast enolase and yeast alcohol dehydrogenase were accompanied by increases in thermal stability at lower extents of modification. Decreases in thermal stability result from greater modification. These results support suggestions that surface guanidino groups (arginyl groups) are an important factor in thermal stability of proteins.     

Positive and negative regulation of T-cell activation by adaptor proteins

Adaptor proteins, molecules that mediate intermolecular interactions, are now known to be as crucial for lymphocyte activation as are receptors and effectors. Extensive work from numerous laboratories has identified and characterized many of these adaptors, demonstrating their roles as both positive and negative regulators. Studies into the molecular basis for the actions of these molecules shows that they function in various ways, including: recruitment of positive or negative regulators into signalling networks, modulation of effector function by allosteric regulation of enzymatic activity, and by targeting other proteins for degradation. This review will focus on a number of adaptors that are important for lymphocyte function and emphasize the various ways in which these proteins carry out their essential roles.     

Amino acid composition and thermal stability of proteins

This study investigates the relationship between the thermal stability of a globular protein and its amino acid composition. The method deals with the relationship between the amino acid compositions and melting points in a set of proteins by computing single-residue and group correlations. Groups of residues are shown to stabilize or destabilize the molecule against temperature. The stabilizing group consists of polar-charged residues and nonpolar residues possessing high surrounding hydrophobicity. The polar-uncharged residues destabilize the molecule against temperature, serine being the most destabilizing residue. A very high cooperativity exists among the stabilizing nonpolar residues suggesting that their characteristic clustering inside the globule may enhance the thermostability of a protein. In small globular proteins which act as single cooperative units, the melting temperature remains mainly a function of amino acid composition, whereas in complex molecules it depends on other factors also.     

Physical and molecular bases of protein thermal stability and cold adaptation

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Role of weak interactions in thermal stability of proteins

A database analysis was done to study the role of weak interactions such as CHcdots, three dots, centeredO, CHcdots, three dots, centeredPI(m) and NHcdots, three dots, centeredPI(m) in the thermal stability of proteins. The CHcdots, three dots, centeredO and CHcdots, three dots, centeredPI(m) interactions are more in the case of thermophilic proteins as compared to mesophiles. Amino acid analysis showed that hydrophobic amino acids like Val and Ile, and Cys contribute more to CHcdots, three dots, centeredO hydrogen bonds where as Pro and Gly contribute more to CHcdots, three dots, centeredPI(m) interactions. Though NHcdots, three dots, centeredPI(m) interactions are dominated by Lys and Arg in thermophiles and mesophiles, the Arg contribution is significantly higher in thermophiles. Interestingly, Glycine is a predominant contributor to all the weak interactions. The number of aromatic amino acids in the thermophiles is more and hence a large number of aromatic clusters were observed in this class. Thus, a cumulative effect of weak interactions seems to be important in thermal stability of proteins. The study also shows that introduction of Gly, Arg, Phe, Pro, and Tyr may enhance the thermal stability.     

Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins

Structural maintenance of chromosomes (SMC) proteins are central regulators of higher-order chromosome dynamics from bacteria to humans. The Bacillus subtilis SMC (BsSMC) homodimer adopts a V-shaped structure with an ATP-binding catalytic domain at each end. We report here that two small proteins, ScpA and ScpB, associate with the catalytic domains of BsSMC in an ordered fashion and suppress its ATPase activity. When combined with a 'transition state' mutant of BsSMC that poorly hydrolyzes ATP, ScpA promotes stable engagement of two catalytic domains in an ATP-dependent manner. In solution, this occurs intramolecularly and closes the DNA-entry gate of an SMC dimer. ScpB further stabilizes this conformation and prevents BsSMC from binding to double-stranded DNA (dsDNA). In contrast, when the mutant BsSMC is first allowed to interact with dsDNA, subsequent addition of ScpA leads to assembly of large nucleoprotein complexes, possibly by stabilizing intermolecular engagement of the catalytic domains from different SMC dimers. We propose that the ATP-modulated engagement/disengagement cycle of SMC proteins plays both positive and negative roles in their dynamic interactions with dsDNA.     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

Microscale fluorescent thermal stability assay for membrane proteins

Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp]. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1093–1099, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature.     

Positive and negative birefringence in chromosomes

By using the optical properties of birefringence of DNA, the arrangement of these molecules has been studied in Dinoflagellate chromosomes and Dipteran polytene chromosomes. These latter are used, here, as a reference material. These observations have been made under a polarizing microscope on intact and stretched chromosomes. — Intact Dinoflagellate chromosomes show a positive birefringence, in contrast with polytene chromosomes bands which are negatively birefringent. From these observations one can deduce the preferential orientation of DNA filaments, in Dinoflagellates, normal to the chromosome axis, and in polytene chromosomes parallel to the same axis. — After stretching, these two kinds of chromosomes are negatively birefringent. In both cases, DNA molecules have been aligned along the stretch axis. — In Dinoflagellate chromosomes the passage from a positive to a negative birefringence is realized without any isotropic stage. The intermediary state presents a biaxial structure.     

InTRIMsic immunity: Positive and negative regulation of immune signaling by tripartite motif proteins

During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties. This is achieved in a process involving E3 ligases which determine ubiquitination target specificity.One of the most prominent E3 ligase families is that of the tripartite motif (TRIM) proteins, which counts over 70 members in humans. Over the last years, various studies have contributed to the notion that many members of this protein family are important immune regulators. Recent studies into the mechanisms by which some of the TRIMs regulate the innate immune system have uncovered important immune regulatory roles of both covalently attached, as well as unanchored poly-ubiquitin chains. This review highlights TRIM evolution, recent findings in TRIM-mediated immune regulation, and provides an outlook to current research hurdles and future directions.     

Increased thermal stability of proteins in the presence of sugars and polyols.

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.     

Shedding light on the extra thermal stability of thermophilic proteins

An entropic stabilization mechanism has recently gained attention and credibility as the physical ground for the extra thermal stability of globular proteins from thermophilic microorganisms. An empirical result, obtained from the analysis of thermodynamic data for a large set of proteins, strengthens the general reliability of the theoretical approach originally devised to rationalize the occurrence of cold denaturation [Graziano, PCCP 2014, 16, 21755–21767]. It is shown that this theoretical approach can readily account for the entropic stabilization mechanism. On decreasing the conformational entropy gain associated with denaturation, the thermal stability of a model globular protein increases markedly.     

The effects of surface adsorption on the thermal stability of proteins

The effect of surface adsorption on the structure and stability of proteins is a matter of increasing interest in biotechnology. Therefore, we have examined the effect of adsorption to silica on the thermal stability of 7 proteins employing differential scanning calorimetry (DSC) and front surface fluorescence (FSF) spectroscopy. In general, it was found that surface adsorption decreased the thermal stability of the bound protein. Using lysozyme for further studies, DSC, FSF, and FTIR spectroscopies, as well as enzymatic activity measurements, were used to explore the effect of decreasing surface apolarity on stability. It was observed that increasing surface apolarity produced decreasing stability and increasing structural alteration of the adsorbed protein.     

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.     

A simple electrostatic criterion for predicting the thermal stability of proteins

The enhancement of protein thermostability is an important issue for both basic science and biotechnology purposes. We have developed a thermostability criterion for a protein in terms of a quasi-electric dipole moment (contributed by its charged residues) defined for an electric charge distribution whose total charge is not zero. It was found that minimization of the modulus of this dipole moment increased its thermal stability, as demonstrated by surveying these values in pairs of mesostable-thermostable homologous proteins and in mutations described in the literature. The analysis of these observations provides criteria for thermostabilization of a protein, by computing its dipole profile. This profile is obtained by direct substitution of each amino acid of the sequence by either a positive, negative or neutral amino acid, followed by a recalculation of the dipole moment. As an experimental example, these criteria were applied to a beta-glucanase to enhance its thermal stability.