Optimized electrostatic surfaces parallel increased thermostability: a structural bioinformatic analysis

It has been known for some time that thermophilic proteins generally have increased numbers of non-covalent interactions (salt bridges, hydrogen bonds, etc.) compared with their mesophilic orthologs. Recently, anecdotal structural comparisons suggest that non-specific acid-base ion pairs on the protein surface can be an evolutionary efficient mechanism to increase thermostability. In this comprehensive structural analysis, we confirm this to be the case. Comparison of 127 orthologous mesophilic- thermophilic protein groups indicates a clear preference for stabilizing acid-base pairs on the surface of thermophilic proteins. Compared with positions in the core, stabilizing surface mutations are less likely to disrupt the tertiary structure, and thus more likely to be evolutionarily selected. Therefore, we believe that our results, in addition to being theoretically interesting, will facilitate identification of charge-altering mutations likely to increase the stability of a particular protein structure.     

Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability.

Understanding the molecular determinants of protein thermostability is of theoretical and practical importance. While numerous determinants have been suggested, no molecular feature has been judged of paramount importance, with the possible exception of ion-pair networks. The difficulty in identifying the main determinants may have been the limited structural information available on the thermostable proteins. Recently the complete genomes for mesophilic, thermophilic and hyperthermophilic organisms have been sequenced, vastly improving the potential for uncovering general trends in sequence and structure evolution related to thermostability and, thus, for isolating the more important determinants. From a comparative analysis of 20 complete genomes, we find a trend towards shortened thermophilic proteins relative to their mesophilic homologs. Moreover, sequence alignments to proteins of known structure indicate that thermophilic sequences are more likely than their mesophilic homologs to have deletions in exposed loop regions. The new genomes offer enough comparable sequences to compute meaningful statistics that point to loop deletion as a general evolutionary strategy for increasing thermostability.     

Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms

MOTIVATION: Understanding the basis of protein stability in thermophilic organisms raises a general question: what structural properties of proteins are responsible for the higher thermostability of proteins from thermophilic organisms compared to proteins from mesophilic organisms? RESULTS: A unique database of 373 structurally well-aligned protein pairs from thermophilic and mesophilic organisms is constructed. Comparison of proteins from thermophilic and mesophilic organisms has shown that the external, water-accessible residues of the first group are more closely packed than those of the second. Packing of interior parts of proteins (residues inaccessible to water molecules) is the same in both cases. The analysis of amino acid composition of external residues of proteins from thermophilic organisms revealed an increased fraction of such amino acids as Lys, Arg and Glu, and a decreased fraction of Ala, Asp, Asn, Gln, Thr, Ser and His. Our theoretical investigation of folding/unfolding behavior confirms the experimental observations that the interactions that differ in thermophilic and mesophilic proteins form only after the passing of the transition state during folding. Thus, different packing of external residues can explain differences in thermostability of proteins from thermophilic and mesophilic organisms. AVAILABILITY: The database of 373 structurally well-aligned protein pairs is available at http://phys.protres.ru/resources/termo_meso_base.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Using a strategy based on the concept of convergent evolution to identify residue substitutions responsible for thermal adaptation

Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration.     

嗜热酶的耐热机理

酶蛋白在高温下的不稳定性是影响其广泛应用的主要瓶颈,嗜热酶因为独特的性质而被作为热稳定研究的极好材料。了解嗜热酶的热稳定性机制,对于采用酶工程定向设计、改造酶具有重要的意义。嗜热酶的热稳定性并不是由单一因素决定的,氨基酸组成、氢键、离子对、二硫键等都是影响嗜热酶热稳定性的重要因素。相对于嗜温酶,嗜热酶更多地采用寡聚体的形式。     

How do thermophilic proteins resist aggregation?

Aggregation is an ancient threat that must be overcome by proteins from all organisms to maintain their native functional states. This is essential for the maintenance of metabolic flux and viability of their cellular machineries. Here, we compare the aggregation-resistance strategies adapted by the thermophilic proteins and their mesophilic homologs using a dataset of 373 protein families. Like their mesophilic homologs, the thermophilic protein sequences also contain potential aggregation prone regions (APRs), capable of forming cross-β motif and amyloid-like fibrils. Tetrapeptide and hexapeptide amyloid-like fibril forming sequence patterns and experimentally proven amyloid-like fibril forming peptide sequences were also detected in the thermophilic proteins. Both the thermophilic and the mesophilic proteins use similar strategies to resist aggregation. However, the thermophilic proteins show superior utilization of these strategies. The thermophilic protein monomers show greater ability to "stow away" the APRs in the hydrophobic cores to protect them from solvent exposure. The thermophilic proteins are also better at gatekeeping the APRs by surrounding them with charged residues (Asp, Glu, Lys, and Arg) and Pro to a greater extent. While thermophilic and mesophilic proteins in our dataset are highly homologous and show strong overall sequence conservation, the APRs are not conserved between the homologs. These findings indicate that evolution is working to avoid amyloidogenic regions in proteins. Our results are also consistent with the observation that thermophilic cells often accumulate small molecule osmolytes capable of stabilizing their proteins and other macromolecules. This study has important implications for rational design and formulation of therapeutic proteins and antibodies.     

氨基酸组成对蛋白质耐热性的影响

为了研究一级结构对蛋白质耐热性的影响,利用软件DNAMAN对16个家族32种蛋白质序列进行了氨基酸含量分析,并统计分析了氨基酸组成对蛋白质耐热性的影响。通过比较同一家族的高低温蛋白质序列及16个家族中所有高温和低温蛋白质序列中氨基酸含量的变化可以推断(从低温到高温)：Ser、Cys．含量降低显著,Arg、Ile、Pro含量升高显著。由此可知高温蛋白质倾向于含有疏水性氨基酸而避免亲水性氨基酸。     

Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.     

Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus.

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.     

Isolation and characterization of a metagenome-derived thermoalkaliphilic esterase with high stability over a broad pH range

A novel alkaliphilic esterase (EstJ) was identified from a soil metagenome of Jeju Island, Korea, using a 96-well plate-based functional assay for determination of pH dependence of activity. The amino acid sequence of EstJ showed low similarity (32–45 %) to putative α/β hydrolases derived from whole-genome sequencing studies. EstJ, although not belonging to any of the known families of bacterial lipolytic enzymes, however, it showed closest sequence identity to the family IV enzymes that are related to the mammalian hormone-sensitive lipases. The highly conserved motifs of family IV enzymes were found in EstJ, but the corresponding sequences of each motif in EstJ were unique; most particularly the –(F/Y)(F/Y/L)HGGG– motif was represented by –WMVSGG–. The purified EstJ was highly active from pH 8.5 to 10.5. More than 90 % of maximum activity was also retained over a wide pH range of 5.5–0.5 after prolonged incubation. EstJ was also moderately thermophilic with an optimum temperature of 55 °C. Therefore, EstJ is the first metagenome-derived bacterial family IV esterase possessing both highly alkaliphilic and moderately thermophilic properties.     

Protein thermostability: structure-based difference of residual properties between thermophilic and mesophilic proteins

Structure-based differences of residual properties between 20 pairs of thermophilic and mesophilic proteins were statistically analyzed to elucidate the factors governing protein thermostability. This study analyzed the distributions of outer residues, inner residues, flexible residues, rigid residues, hydrogen bonds, salt bridges, cation–pi interactions, and disulfide bonds in each protein in terms of residual structural states, which were determined as five kinds of states under residual packing value. Their structural patterns found in thermophilic protein groups were compared with those of mesophilic protein groups for showing distinctive difference of residual properties. The results of statistical tests (t-test) revealed that flexible residues in fully-exposed state and boundary state, salt bridges in exposed state, and hydrogen bonds in well-buried state could be critical factors related with protein thermostability. Such structure-based differences of residual properties would help to develop a strategy for enhancing protein thermostability.     

Robust determinants of thermostability highlighted by a codon frequency index capable of discriminating thermophilic from mesophilic genomes

Can genome analysis tell us about the lifestyle of an organism? We ask this question considering a thorough cross comparison of thermophilic and mesophilic genomes, since presently the number of available genomes is enough to ensure statistical significance of the results. We analyze, by means of principal component analysis (PCA), the codon composition of a database comprising 116 genomes, selected so as to include one species for each genus and show that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level. The results of our analysis indicate that all the known features of thermostability can be found in the 64 component loadings of the second principal axis of PCA. By this, we develop an index of thermostability whose discriminative power between mesophiles and thermophiles scores with 98% accuracy at the genome level and with 95% accuracy at the protein sequence level. We also prove that these results are not due to phylogenetic differences between archaea and bacteria.     

The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.     

Modularity of the slit protein. Characterization of a conserved carboxy-terminal sequence in secreted proteins and a motif implicated in extracellular protein interactions.

Since our characterization of the slit cDNA sequence, encoding a protein secreted by glial cells and involved in the formation of axonal pathways in Drosophila, we have discovered that the protein contains two additional sequence motifs that are highly conserved in a variety of proteins. A search of the GenPept database with the 73 amino acids at the carboxy terminus of slit revealed that this region contains significant similarity to a carboxy-terminal domain found in six other exported proteins. This observation has allowed us to define a new carboxy-terminal protein motif. In addition, comparisons with a 202 amino acid domain residing between epidermal growth factor (EGF) repeats in slit shows this region to be conserved in laminin, agrin and perlecan and, strikingly, also to lie between EGF repeats in both agrin and perlecan. Our analysis suggests this motif is involved in mediating interactions among extracellular proteins. Consistent with our previous characterization of the slit protein, both new motifs are found only in extracellular proteins. The identification of these two conserved motifs in slit reveals that the entire 1469 amino acids of the protein are made up of modular regions similar to those conserved in other extracellular proteins.     

Distance-dependent statistical potentials for discriminating thermophilic and mesophilic proteins

Identification of the characteristic structural patterns responsible for protein thermostability is theoretically important and practically useful but largely remains an open problem. These patterns may be revealed through comparative study on thermophilic and mesophilic proteins that have distinct thermostability. In this study, we constructed several distance-dependant potentials from thermophilic and mesophilic proteins. These potentials were then used to evaluate the structural difference between thermophilic and mesophilic proteins. We found that using the subtraction or division of the potentials derived from thermophilic and mesophilic proteins can dramatically increase the discriminatory ability. This approach revealed that the ability to distinct the subtle structural features responsible for protein thermostability may be effectively enhanced through rationally designed comparative study.