- Research article
- Open Access
Consistent mutational paths predict eukaryotic thermostability
© van Noort et al.; licensee BioMed Central Ltd. 2013
- Received: 31 August 2012
- Accepted: 7 January 2013
- Published: 10 January 2013
Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published.
Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations.
The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.
- Comparative genomics
- Protein engineering
Proteins from thermophilic organisms are not only stable at higher temperatures, but are also generally more stable than their mesophilic counterparts. Therefore they are scientifically valuable, e.g. for biochemical and structural studies, and have multiple applications in industry . However, many proteins exclusively occur in eukaryotes, and only a few of the latter are thermophilic (defined as having an optimal growth temperature [OGT] above 50°C; . Recently, the first eukaryotic thermophilic genome, Chaetomium thermophilum, gave first insights into the potential for structural biology . Now with two more genomes, Thielavia terrestris and Thielavia heterothallica being published, comparative analysis of their thermophilic nature can be performed.
Thielavia terrestris and Thielavia heterothallica (anamorph Myceliophtora thermophila) are filamentous fungi of the class Sordariomycetes  which can be found in ‘unnatural’ habitats like compost. Their natural habitat seems to be in soils such as in semi-arid grasslands in New Mexico . They are common in multiple microhabitats in this region, where high summer temperatures in combination with episodes of substantial precipitation provide favourable conditions . Chaetomium thermophilum is a widely distributed soil-inhabiting fungus and a thermophile in accordance with its lifestyle in self-heating composting plant material . It can also be found in composting urban solid waste [7, 8] and wood-chip piles [9, 10]. C. thermophilum is a member of the large genus of Chaetomium, also within the Sordariomycetes, that are found in soil, air, and plant debris . Close relatives of these thermophilic fungi are the mesophilic mould fungus Chaetomium globosum (OGT 24°C), a frequent indoor contaminant that produces mycotoxins and acts as an allergen , and Neurospora crassa, another mesophilic filamentous fungus of which the genome has been published .
Due to their thermostable nature, proteins from thermophilic fungi have recently gained considerable attention in industry and structural biology. Several crystal structures of proteins from these thermophilic fungi have been determined such as those of two beta 1,4-galactanases from T.heterothallica, a glycoside hydrolase from T. terrestris, and Get3, Get4 and beta 1,4-xylanase from C. thermophilum[15–17]. The paper-industry utilizes members of the beta 1,4-xylanase family for bio-bleaching of kraft-pulp [18, 19]. The biotechnological potential of C. thermophilum is also illustrated by the purification and characterization of its thermostable superoxide dismutase (SOD) , an enzyme which is utilized in cosmetic products to reduce free radical damage to the skin. Furthermore, the genomes of C. thermophilum, T. terrestris and T. heterothallica provide a source of thermostable cellulolytic enzymes, such as the glycoside hydrolases that can be used in the production of third-generation biofuels .
Here, we identify commonalities and differences of thermophilic adaptation between eukaryotes and prokaryotes and exploit the close relationship of the thermophilic to mesophilic fungi to gain detailed insight into the molecular evolution of thermophily. By comparing the genomes of thermophilic fungi to each other and to mesophilic relatives we can clarify the evolutionary trajectory that has been obscured by inconsistent naming conventions  and determine whether there are independent events of gain of thermophily in these fungi. We further use the observed adaptation biases to predict mutations that can increase the thermostability of proteins and verify them experimentally.
Taxonomic position of thermophilic fungi within Chaetomiaceae
Genome reduction in thermophiles
The genomes of C. thermophilum (Cth), T. terrestris (Tte) and T. heterothallica (Tht) are significantly smaller than their close mesophilic relatives such as Chaetomium globosum (Cgl) and Neurospora crassa (Ncr). In agreement with previous studies of prokaryotic thermophiles, the genome size reduction is due mainly to fewer protein coding genes (Cth 7,267; Tte 9,813; Tht 9,110 vs Cgl 11,124 and Ncr 10,620), but also to shorter introns and shorter intergenic regions (Additional file 1: Figure S2) and . Since C. globosum is derived from the ancestor of these three species, there are two possibilities. Either, this ancestor had a small genome and C. globosum has gained genes by duplications or horizontal transfers or the three thermophiles have independently lost genes in a parallel adaptation process. Although larger genomes in the outgroups makes a loss and gain scenario more likely, we investigated all orthologous groups from the complete genomes of 20 members of the Sordariomycetes (sorNOGs) to clarify the gene content evolution of eukaryotic thermophiles. Firstly, we analysed the phylogenetic presence/absence patterns of these sorNOGs. In total, 4,542 protein coding genes are present in equal copy numbers in each of the four species Cth, Tte, Tht and Cgl. Present in one copy but absent from either of the four are 330 (Cgl), 125 (Tte), 130 (Tht) and 440 (Cth) orthologs, meaning that lineage specific loss alone does not account for the differences in genome size. C. globosum specific duplications are responsible for ca. 150 extra genes. It must be noted that some lineage specific losses may also be accounted for by difference in genome quality, but the tendencies will remain.
On the other hand, there are 845 orthologous groups covering 1,004 genes of C. globosum that are absent in all three others. These numbers are 181 (190), 325 (353) and 543 (579) orthologous groups (genes) for Cth, Tte and Tht. The difference in genome size can thus partly be assigned to these orthologous groups. A large number of these are related to transposable elements, including 30 transposases, 74 reverse transcriptases, 30 DNA helicases. The lack of these elements in the thermophilic fungi may indicate that transposition is unfavourable at higher temperatures.
Oxygenases and enzymes hydrolyzing complex sugars are in particular frequently lost in the thermophiles. This does not always mean that metabolic capabilities are completely absent; often multigene families in N. crassa and C. globosum have only one counterpart in C. thermophilum, but also non-homologous isoforms are reduced to one enzyme, implying a reduction in robustness. Proteins that are completely missing in C. thermophilum but not in the two Thielavias include WC1, WC2 and FRQ which are involved in the regulation of the circadian clock [23, 24]. We hypothesize that due to the localization far inside the compost away from light (implied by the high temperature optimum) the day-night rhythm does not play a role for C. thermophilum.
There are no major gene family expansions in the thermophiles compared to their relatives, only a few orthologous groups have been slightly expanded against the reductionist trend. The majority of them are uncharacterized, but some indicate life style adaptation such as a cellobiose dehydrogenase of which C. thermophilum has three copies and C. globosum and N. crassa only two, reflecting an increased wood degradation capacity. T. terrestris has five copies of a S-adenosyl-L-methionine (SAM) dependent methyltransferase that is likely to employ arsenite as substrate where its relatives have only one or two. The largest lineage specific expansion in T. heterothallica is an orthologous group with three copies of a scytalone dehydratase involved in fungal melanin biosynthesis. Melanin provides resistance to UV radiation, drought and high temperatures  and thus this expansion likely represents a thermophilic adaptation. The lack of major expansions suggests that the metabolisms of the thermophilic fungi have not undergone major niche adaptations requiring additional functionality, and that the dominating adaptation was indeed the one to higher temperatures.
Convergent evolution of thermophily across all domains of life
It has been previously shown that the amino acid frequencies vary with the OGT, specifically the summed frequency of the amino acids IVYWREL shows the highest correlation with OGT in both bacteria and archaea . In these domains of life, the ancestor was likely a thermophile and adaptation happened to colder environments .
Genomes used for amino acid bias analysis
Coccidioides immitis h538
Coccidioides immitis rs
Histoplasma capsulatum h143
Histoplasma capsulatum h88
Analyzing the amino acid frequencies from bacterial (Additional file 1: Table S1) and archaeal (Additional file 1: Table S2) clades with thermophilic members, we observe a striking difference with eukaryotes; an overrepresentation of cysteines in C. thermophilum proteins (Figure 1B); in total 15% of cysteines in aligned positions are unique to C. thermophilum. The major categorized roles of cysteines are in catalytic residues, disulfide bridges and metal binding (e.g. zinc fingers), whereby the latter two contribute to folding and stability. Cysteines have also been shown to contribute to thermal stability in their free form, when they form interactions inside the core of a protein . This unique adaptation of C. thermophilum may be another indication that its proteins are better adapted to high temperatures than the other two thermophilic Sordariomycetes. Another difference between prokaryotes and eukaryotes that we observe is that glycines are strongly depleted in C. thermophilum whereas they are enriched in C. globosum compared to the complete clade of Sordariomycetes. The exchange of alanines with glycines has been shown to destabilize alpha-helices, particularly in the center of the helix . It seems as if C. globosum has indeed used this strategy to make proteins less thermo-stable, and C. thermophilum has evolved in the opposite direction, lowering its glycine content.
We verified the generalizability of these trends by examining two more unpublished thermophilic fungal genomes, Thermomyces lanuginosus and Talaromyces thermophilus of the subclass Eurotiomycetidae, a different fungal clade that also includes Aspergillus fumigatus and Emericella nidulans. Compared to their mesophilic neighbours, these species both have a significantly higher total frequency of IVYWREL amino acids (P < 1e-7). They also show a depletion of glycines and significant enrichment in arginines and alanines (Figure 1C) consistent with the biases in the thermophilic Sordariomycetes. This shows that some of the trends are indeed universal between different clades of fungi.
Mutational paths towards thermophily
Considering the consistent biases, we analysed particular residues in orthologous groups shared between Sordariomycetes and Eurotiomycetidae. We found that where the same biases exist, the overlap between positions where e.g. arginines have been introduced instead of lysines is significant but small, i.e. 92 out of a total of 7,335 positions that are changed from lysine to arginine in any of the thermophiles are shared between all five. This leads us to believe there are some positions that are more likely to increase stability and may even be essential; however mutations at many other positions can contribute independently.
In contrast to prokaryotes where GC content has been found to cause a bias in amino acid frequencies of lysines and arginines [35, 36], as previously reported in these fungi the GC content does not differ significantly between mesophiles and thermophiles . There is an elevated GC content at the third codon position as reported by , however the frequencies of G at C and the third codon position do not differ between lysine and arginine. Therefore, in thermophilic fungi the lysine-arginine bias has arisen independently of the GC content.
Scoring scheme for adaptive mutations
Based on our observations, we developed a scoring scheme to give weight to individual mutations for their contribution to thermophily (Figure 2). We used the mutation bias between pairs of amino acids in the branches leading to the thermophilic ancestor as well as to C. thermophilum to arrive at these scores (see Methods). We predict that those positions with a high score are responsible for the thermophilic adaptation of individual proteins. In this way, we can distinguish which thermophile specific mutations are likely to be adaptive and which are likely to be neutral. Since the thermophilic nature of proteins has been lost in C. globosum, we can also predict which mutations have been responsible for this loss. In this way we predicted 38,385 thermophilic adaptive mutations in 2,064 single copy proteins for which we could trace the ancestral amino acid sequences.
Mutations important for thermophilic stability
Structural context for adaptive mutations
Here, we show that the principles of thermophilic adaptations in fungi are similar to that in prokaryotes, with the notable exception of cysteines that are enriched in C. thermophilum and that might contribute to thermophily in several ways. The close relation of mesophilic species allows predicting particular mutations that are directly responsible for thermo-adaptation, which we could confirm experimentally by protein engineering. By solving the 3D structure of a single thermophilic protein (Arx1 of Cth), we could identify three different types of adaptive mutations : (i) loop rigidity by increased proline frequency, (ii) increased protein core hydrophobicity, and (iii) increased electrostatic interactions stabilizing neighboring secondary structure elements.
By now, several structures have been determined already based on C. thermophilum, T. terrestris and T. heterothallica proteins [13–17] and we and others have determined the thermostable nature of several other proteins [3, 41]. This, together with our finding of thousands of mutations towards thermophily in this lineage, implies that the thermostability of proteins is a major contributor to the increased OGT of these organisms, in particular in C. thermophilum. C. thermophilum is, as are T. terrestris and T. heterothallica promising resources for (thermo)stable proteins for industrial purposes as well as for biochemical and structural studies that rely on stable eukaryotic proteins and the assembly of complex molecular machines. With experimental tools such as genetic transformation protocols and a number of independent lineages containing thermophilic eukaryotes, a rapidly increased understanding should lead to precise predictions which particular mutation increases thermophily via which mechanisms for a vast amount of important eukaryotic proteins.
Fungal orthologous groups
Published genomes were downloaded from NCBI. Unpublished genomes were downloaded from ftp-sites of the Joint Genome Institute, the BROAD institute and Genome Canada. Non-supervised orthologous groups (NOGs) were constructed for 20 Sordariomycetes and 21 Eurotiomycetidae (Table 1) through identification of reciprocal best BLAST  matches and triangular linkage clustering as implemented in eggNOG v2 . This resulted for Sordariomycetes in 17,325 and for Eurotiomycetidae in 14,979 non-supervised orthologous groups (NOGs). Out of 7,227 C. thermophilum proteins, we find orthologs in other Sordariomycetes for 7,045 of them. We found 2,064 NOGs that contain exactly one copy from each Sordariomycetes proteome (universal single copy orthologs) and 1,436 that contain exactly one copy from each of the Eurotiomycetidae. HMMs of 40 marker genes  were used to search all 20 Sordariomycetes proteomes. These were aligned using MUSCLE , pruned with GBLOCKS  and a tree was built using RAxML . Trees were displayed using iTOL . The same procedure was applied for all 2,064 universal single copy orthologs.
Amino acid overrepresentation
Alignments of universal single copy orthologs were made using MUSCLE  with standard settings. Amino acid frequencies were counted in all aligned positions that do not contain gaps. The frequencies in C. thermophilum were compared against frequencies in mesophilic Sordariomycetes and Z-tests were done to obtain significant differences in all aligned positions. Similarly, T-tests were done to obtain significant differences between the three thermophiles and their ancestral nodes on the one hand and the mesophiles and their ancestral nodes on the other hand. Significant differences are shown as stars in Figure 1B and C. A similar analysis was done in a group of seven bacteria (Additional file 1: Table S1) and in a group of seven archaea (Additional file 1: Table S2) with large variance in the optimal growth temperatures. In this case there was not one species that was thermophilic, therefore, rather than a Z-test, correlations of the AA-frequencies with OGT were calculated (Additional file 1: Table S3) and t-tests were done between the AA-frequencies of (hyper)thermophilic and mesophilic species to obtain significances (Additional file 1: Table S3).
Parsimonious reconstructions of ancestral states were made using PROTPARS from the Phylip package  with the fungi tree as user tree for all single copy NOG alignments. From the output file, steps at each position were parsed and counted only if they were unambiguous. The frequencies of mutations between all pairs of amino acids were analysed. The ratios between all pairs of amino acids were compared to the ratios in the whole reconstructed phylogeny. In principle, it is expected that there are as many mutations from X to Y as from Y to X. Thus a bionomial test can be used to assess a bias. However, there are also biases in the complete groups of Sordariomycetes and Eurotiomycetidae. Therefore the expected ratio is not set to 1:1, but to the actual ratio in the mesophilic neighboring species.
Scoring of amino acid substitutions
We developed a scoring scheme to give a weight to individual mutations for their contribution to thermophily. We used the mutation bias between pairs of amino acids to arrive at these scores. We calculate the binomial probability of the number of mutations from amino acid X to Y vs Y to X, given the average ratio between X to Y and Y to X in the whole Sordariomycetes tree. The logarithm of this probability is multiplied by −1 to come to a score S for pair X and Y. If in a phylogenetic reconstruction, there is a mutation from X to Y and there is a significant bias from X to Y, this mutation will get the positive score S, if there is a significant bias from Y to X, it will get the negative score S, otherwise the mutation is not scored.
Purification of recombinant protein
ORFs for cgARX1, ctARX1, ctarx1-destab and ctarx1-nondestab were synthesized and sequenced by Eurofins MWG Operon (Ebersberg, Germany) or GenScript (Piscataway, NJ, USA) and subcloned into pET-24a(+) vector. Proteins were expressed in E. coli BL21 (DE3) grown in LB-medium at 37°C under vigorous shaking. Cell pellets were resuspended in buffer A (20 mM Hepes-NaOH pH 8.0, 350 mM NaCl, 10 mM KCl, 10 mM MgCl2, 40 mM imidazol). Cells were lysed by a Microfluidizer (M-110 L, Microfluidics) and the lysate was cleared by ultracentrifugation at 91,000 × g for 20 minutes. Recombinant protein was purified by Ni-ion affinity chromatography (Ni-NTA-HisTrap, GE-Healthcare) via an N-terminal hexa-histidine tag and eluted with buffer A supplemented with 460 mM imidazol. ctArx1 was further purified by size exclusion chromatography (S200-26/60, GE-Healthcare) in a buffer containing 20 mM Hepes-NaOH pH 8.0, 200 mM NaCl, 10 mM KCl and 10 mM MgCl2.
Crystallization and structure determination of ctArx1
Crystal data of Arx1 from C. thermophilum
Unit cell parameters (Å)
192.0, 193.3, 70.9
90, 90, 90
86.4 – 2.3 (2.42 – 2.3)
Number of used reflections
Resolution limits (Å)
R factor b (%)
Free R factor c (%)
Rmsd bond lengths (Å)
Rmsd bond angles (°)
Thermostabilites of ctArx1 and cgArx1 were determined by testing an in vitro aggregation. For this assay, recombinant ctArx1 and cgArx1 were purified from E. coli and incubated at the indicated temperatures (see Figure 3B, D, Additional file 1: Figure S7B) for one hour in buffer 2 (50 mM Tris–HCl pH 7.5, 200 mM NaCl, 10 mM KCl, 10 mM MgCl2, 5% (v/v) glycerol, 0.01% (v/v) MTG). Following centrifugation at 20,000 rpm at 4°C for 30 minutes, an equivalent sample of the supernatant and the pellet fraction was separated by SDS-polyacrylamide gel electrophoresis (PAGE; NuPAGE 4–12% Bis-Tris Gel, Invitrogen). Proteins were visualized with Coomassie (Brilliant Blue G – colloidal Concentrate, Sigma-Aldrich).
Thermal unfolding transitions of ctArx1 were followed by circular dichroism at 222.6 nm with 1 nm bandwidth in 2 mm cells and a heating rate of 1°C per minute using a Jasco J-810 spectropolarimeter in 10 mM potassium phosphate, pH 7.5, at a protein concentration of ~0.1 mg/ml.
We thank Christian von Mering for help using the eggNOG pipeline. E.H. is recipient of grants from the Deutsche Forschungsgemeinschaft (SFB 638/B2). The structure of the ctArx1 protein has been submitted to the PDB with accession number 4IPA. The Tree presented in Figure 1 is available at TreeBASE with accession number 13595.
- Wimberly BT, Brodersen DE, Clemons WM, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V: Structure of the 30S ribosomal subunit. Nature. 2000, 407 (6802): 327-339. 10.1038/35030006.PubMedView ArticleGoogle Scholar
- Hickey DA, Singer GA: Genomic and proteomic adaptations to growth at high temperature. Genome Biol. 2004, 5 (10): 117-10.1186/gb-2004-5-10-117.PubMed CentralPubMedView ArticleGoogle Scholar
- Amlacher S, Sarges P, Flemming D, van Noort V, Kunze R, Devos DP, Arumugam M, Bork P, Hurt E: Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell. 2011, 146 (2): 277-289. 10.1016/j.cell.2011.06.039.PubMedView ArticleGoogle Scholar
- Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A: Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol. 2011, 29 (10): 922-927. 10.1038/nbt.1976.PubMedView ArticleGoogle Scholar
- Powell AJ, Parchert KJ, Bustamante JM, Ricken B, Hutchinson MI, Natvig DO: Thermophilic fungi in an aridland ecosystem. Mycologia. 2012, 104 (4): 813-825. 10.3852/11-298.PubMedView ArticleGoogle Scholar
- Chang Y, Hudson HJ: The fungi of wheat straw compost: I. Ecological studies. Trans Br Mycol Soc. 1967, 50 (4): 649-666. 10.1016/S0007-1536(67)80097-4.View ArticleGoogle Scholar
- de Bertoldi M, Vallini G, Pera A: The biology of composting: A review. Waste Manag Res. 1983, 1 (2): 157-176. 10.1016/0734-242X(83)90055-1.View ArticleGoogle Scholar
- Kane BE, Mullins JT: Thermophilic fungi in a municipal waste compost system. Mycologia. 1973, 65 (5): 1087-1100. 10.2307/3758290.PubMedView ArticleGoogle Scholar
- Greaves H: Microbiological aspects of wood chip storage in tropical environments. Aust J Biol Sci. 1975, 28 (3): 323-330.PubMedGoogle Scholar
- Tansey M: Isolation of thermophilic fungi from self-heated, industrial wood chip piles. Mycologia. 1971, 63 (3): 537-547. 10.2307/3757550.View ArticleGoogle Scholar
- Figueras MJ, Cano JF, Guarro J: Ultrastructural alterations produced by sertaconazole on several opportunistic pathogenic fungi. J Med Vet Mycol. 1995, 33 (6): 395-401. 10.1080/02681219580000761.PubMedView ArticleGoogle Scholar
- Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B: The genome sequence of the filamentous fungus Neurospora crassa. Nature. 2003, 422 (6934): 859-868. 10.1038/nature01554.PubMedView ArticleGoogle Scholar
- Le Nours J, Ryttersgaard C, Lo Leggio L, Ostergaard PR, Borchert TV, Christensen LL, Larsen S: Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum. Protein Sci. 2003, 12 (6): 1195-1204. 10.1110/ps.0300103.PubMed CentralPubMedView ArticleGoogle Scholar
- Harris PV, Welner D, McFarland KC, Re E, Navarro Poulsen JC, Brown K, Salbo R, Ding H, Vlasenko E, Merino S, Xu F, Cherry J, Larsen S, Lo Leggio L: Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry. 2010, 49 (15): 3305-3316. 10.1021/bi100009p.PubMedView ArticleGoogle Scholar
- Hakulinen N, Turunen O, Janis J, Leisola M, Rouvinen J: Three-dimensional structures of thermophilic beta-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. Comparison of twelve xylanases in relation to their thermal stability. Eur J Biochem. 2003, 270 (7): 1399-1412. 10.1046/j.1432-1033.2003.03496.x.PubMedView ArticleGoogle Scholar
- Bozkurt G, Stjepanovic G, Vilardi F, Amlacher S, Wild K, Bange G, Favaloro V, Rippe K, Hurt E, Dobberstein B, Sinning I: Structural insights into tail-anchored protein binding and membrane insertion by Get3. Proc Natl Acad Sci USA. 2009, 106 (50): 21131-21136. 10.1073/pnas.0910223106.PubMed CentralPubMedView ArticleGoogle Scholar
- Bozkurt G, Wild K, Amlacher S, Hurt E, Dobberstein B, Sinning I: The structure of Get4 reveals an alpha-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis. FEBS Lett. 2010, 584 (8): 1509-1514. 10.1016/j.febslet.2010.02.070.PubMedView ArticleGoogle Scholar
- Ghaffar A, Khan SA, Mukhtar Z, Latif F, Rajoka MI: Optimized expression of a thermostable xylanase 11 a gene from Chaetomium thermophilum NIBGE 1 in Escherichia coli. Protein Pept Lett. 2009, 16 (4): 356-362. 10.2174/092986609787848126.PubMedView ArticleGoogle Scholar
- Mantyla A, Paloheimo M, Hakola S, Lindberg E, Leskinen S, Kallio J, Vehmaanpera J, Lantto R, Suominen P: Production in trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp. Appl Microbiol Biotechnol. 2007, 76 (2): 377-386. 10.1007/s00253-007-1020-y.PubMedView ArticleGoogle Scholar
- Guo FX, Shi-Jin E, Liu SA, Chen J, Li DC: Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum. Mycologia. 2008, 100 (3): 375-380. 10.3852/06-111R.PubMedView ArticleGoogle Scholar
- Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P: Toward automatic reconstruction of a highly resolved tree of life. Science. 2006, 311 (5765): 1283-1287. 10.1126/science.1123061.PubMedView ArticleGoogle Scholar
- Muller J, Szklarczyk D, Julien P, Letunic I, Roth A, Kuhn M, Powell S, von Mering C, Doerks T, Jensen LJ, Bork P: eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced non-supervised orthologous groups, species and functional annotations. Nucleic Acids Res. 2010, 38 (Database issue): D190-195.PubMed CentralPubMedView ArticleGoogle Scholar
- Schwerdtfeger C, Linden H: Localization and light-dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in Neurospora crassa. Eur J Biochem. 2000, 267 (2): 414-422. 10.1046/j.1432-1327.2000.01016.x.PubMedView ArticleGoogle Scholar
- Aronson BD, Johnson KA, Dunlap JC: Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation. Proc Natl Acad Sci USA. 1994, 91 (16): 7683-7687. 10.1073/pnas.91.16.7683.PubMed CentralPubMedView ArticleGoogle Scholar
- Gao Q, Garcia-Pichel F: Microbial ultraviolet sunscreens. Nat Rev Microbiol. 2011, 9 (11): 791-802. 10.1038/nrmicro2649.PubMedView ArticleGoogle Scholar
- Zeldovich KB, Berezovsky IN, Shakhnovich EI: Protein and DNA sequence determinants of thermophilic adaptation. PLoS Comput Biol. 2007, 3 (1): e5-10.1371/journal.pcbi.0030005.PubMed CentralPubMedView ArticleGoogle Scholar
- Hummer G, Garde S, Garcia AE, Paulaitis ME, Pratt LR: The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. Proc Natl Acad Sci USA. 1998, 95 (4): 1552-1555. 10.1073/pnas.95.4.1552.PubMed CentralPubMedView ArticleGoogle Scholar
- Sandgren M, Gualfetti PJ, Paech C, Paech S, Shaw A, Gross LS, Saldajeno M, Berglund GI, Jones TA, Mitchinson C: The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability. Protein Sci. 2003, 12 (12): 2782-2793. 10.1110/ps.03220403.PubMed CentralPubMedView ArticleGoogle Scholar
- Chakrabartty A, Schellman JA, Baldwin RL: Large differences in the helix propensities of alanine and glycine. Nature. 1991, 351 (6327): 586-588. 10.1038/351586a0.PubMedView ArticleGoogle Scholar
- McDonald JH: Temperature adaptation at homologous sites in proteins from nine thermophile-mesophile species pairs. Genome Biol Evol. 2010, 2: 267-276. 10.1093/gbe/evq017.PubMed CentralPubMedView ArticleGoogle Scholar
- Matthews BW, Nicholson H, Becktel WJ: Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA. 1987, 84 (19): 6663-6667. 10.1073/pnas.84.19.6663.PubMed CentralPubMedView ArticleGoogle Scholar
- Arnorsdottir J, Sigtryggsdottir AR, Thorbjarnardottir SH, Kristjansson MM: Effect of proline substitutions on stability and kinetic properties of a cold adapted subtilase. J Biochem. 2009, 145 (3): 325-329. 10.1093/jb/mvn168.PubMedView ArticleGoogle Scholar
- Hardy F, Vriend G, Veltman OR, van der Vinne B, Venema G, Eijsink VG: Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett. 1993, 317 (1–2): 89-92.PubMedView ArticleGoogle Scholar
- Cupo P, El-Deiry W, Whitney PL, Awad WM: Stabilization of proteins by guanidination. J Biol Chem. 1980, 255 (22): 10828-10833.PubMedGoogle Scholar
- Kreil DP, Ouzounis CA: Identification of thermophilic species by the amino acid compositions deduced from their genomes. Nucleic Acids Res. 2001, 29 (7): 1608-1615. 10.1093/nar/29.7.1608.PubMed CentralPubMedView ArticleGoogle Scholar
- Lobry JR, Chessel D: Internal correspondence analysis of codon and amino-acid usage in thermophilic bacteria. J Appl Genet. 2003, 44 (2): 235-261.PubMedGoogle Scholar
- Nissan TA, Bassler J, Petfalski E, Tollervey D, Hurt E: 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J. 2002, 21 (20): 5539-5547. 10.1093/emboj/cdf547.PubMed CentralPubMedView ArticleGoogle Scholar
- Thompson JD, Gibson TJ, Higgins DG: Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics. 2002, 2.3.1-2.3.22.Google Scholar
- Lowther WT, Matthews BW: Structure and function of the methionine aminopeptidases. Biochim Biophys Acta. 2000, 1477 (1–2): 157-167.PubMedView ArticleGoogle Scholar
- Kowalinski E, Bange G, Bradatsch B, Hurt E, Wild K, Sinning I: The crystal structure of Ebp1 reveals a methionine aminopeptidase fold as binding platform for multiple interactions. FEBS Lett. 2007, 581 (23): 4450-4454. 10.1016/j.febslet.2007.08.024.PubMedView ArticleGoogle Scholar
- Wang XJ, Peng YJ, Zhang LQ, Li AN, Li DC: Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum. Appl Microbiol Biotechnol. 2012, 95 (6): 1469-1478. 10.1007/s00253-011-3799-9.PubMedView ArticleGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedView ArticleGoogle Scholar
- Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32 (5): 1792-1797. 10.1093/nar/gkh340.PubMed CentralPubMedView ArticleGoogle Scholar
- Castresana J: Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000, 17 (4): 540-552. 10.1093/oxfordjournals.molbev.a026334.PubMedView ArticleGoogle Scholar
- Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006, 22 (21): 2688-2690. 10.1093/bioinformatics/btl446.PubMedView ArticleGoogle Scholar
- Letunic I, Bork P: Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2007, 23 (1): 127-128. 10.1093/bioinformatics/btl529.PubMedView ArticleGoogle Scholar
- Felsenstein J: Phylip - phylogeny inference package (version 3.2). Cladistics. 1989, 5: 164-166.Google Scholar
- Bailey S: THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY. ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY. 1994, 50 (5): 760-763. 10.1107/S0907444994003112.View ArticleGoogle Scholar
- Read RJ: Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr D Biol Crystallogr. 2001, 57: 1373-1382. 10.1107/S0907444901012471.PubMedView ArticleGoogle Scholar
- Emsley P, Cowtan K: Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004, 60 (Pt 12 Pt 1): 2126-2132.PubMedView ArticleGoogle Scholar
- Murshudov GN, Vagin AA, Dodson EJ: Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997, 53: 240-255. 10.1107/S0907444996012255.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.