Dynamics of endoglucanase catalytic domains: implications towards thermostability

Thermostable endoglucanases play a crucial role in the production of biofuels to breakdown plant cellulose. Analyzing their structure-dynamics relationship can inform about the origins of their thermostability. Although tertiary structures of many endoglucanase proteins are available, the relationship between thermostability, structure, and dynamics is not explored fully. We have generated elastic network models for thermostable and mesostable endoglucanases with the (αβ)? fold in substrate bound and unbound states. The comparative analyses shed light on the relation between protein dynamics, thermostability, and substrate binding. We observed specific differences in the dynamic behavior of catalytic residues in slow modes: while both the nucleophile and the acid/base donor residues show positively correlated motions in the thermophile, their dynamics is uncoupled in the mesophile. Our proof-of-concept comparison study suggests that global dynamics can be harnessed to further our understanding of thermostability.     

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.     

残基突变对冷休克蛋白热稳定性影响的计算分析研究(英文)

残基突变是提高蛋白质热稳定性最直接有效的方式。在本文中,我们选取一对冷休克蛋白质作为研究对象,其中一个来自嗜温的Bacillus subtilis(Bs-CspB),另一个来自嗜热的Bacillus caldolyticus(Bc-Csp),这两个蛋白质在序列和结构上具有高度的相似性,但两者的耐热能力却相差很大。我们利用全原子模型计算残基突变前后蛋白质的自由能和氨基酸之间相互作用能的变化,分析残基突变对冷休克蛋白热稳定性的影响。通过对比两个蛋白质对应位置上残基的能量,我们成功鉴别出对Bc-Csp的高热稳定性有突出贡献的残基。我们计算了这些残基突变前后,该残基的静电相互作用和范德华相互作用的变化,以分析该残基对Bc-Csp高热稳定性的主要贡献。同时,我们分析了离子键对蛋白质热稳定性的贡献。我们的计算结果和实验结果吻合得很好,关键在于利用该方法可以详细地说明残基突变影响蛋白质热稳定性的根本原因。本文为研究残基突变对蛋白质热稳定性的影响提供了一种计算思路和方法,并有助于设计具有高耐热能力的蛋白质。     

Temperature dependent dynamics in highly homologous adenylate kinases

To correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.     

The effects of ionic strength on protein stability: the cold shock protein family

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.     

Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins

Understanding the role of various interactions in enhancing the thermostability of proteins is important not only for clarifying the mechanism of protein stability but also for designing stable proteins. In this work, we have analyzed the thermostability of 16 different families by comparing mesophilic and thermophilic proteins with 48 various physicochemical, energetic and conformational properties. We found that the increase in shape, s (location of branch point in side chain) increases the thermostability, whereas, an opposite trend is observed for Gibbs free energy change of hydration for native proteins, GhN, in 14 families. A good correlation is observed between these two properties and the simultaneous increases of -GhN and s is necessary to enhance the thermostability from mesophile to thermophile. The increase in shape, which tends to increase with increasing number of carbon atoms both for polar and non-polar residues, may generate more packing and compactness, and the position of beta and higher order branches may be important for better packing. On the other hand, the increase in -GhN in thermophilic proteins increases the solubility of the proteins. This tendency counterbalances the increases in insolubility and unfolding heat capacity change due to the increase in the number of carbon atoms. Thus, the present results suggest that the stability of thermophilic proteins may be achieved by a balance between better packing and solubility.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Purification and characterization of Bacillus coagulans oligo-1,6-glucosidase

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity. The relative molecular mass, Stokes radius, sedimentation coefficient at 20 degrees C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 X 10(-13) s, 1.34 X 10(5) M-1 cm-1, and 4.3, respectively. The amino-terminal amino acid was threonine. There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile). These oligo-1,6-glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile----facultative thermophile----obligate thermophile enzymes.     

The universal ancestor was a thermophile or a hyperthermophile.

By exploiting the correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter thermophile/hyperthermophile proteins, an analysis is conducted in order to establish whether the last universal common ancestor (LUCA) was a mesophile or a (hyper)thermophile. This objective is reached by using maximum parsimony and maximum likelihood to reconstruct the ancestral sequences of the LUCA for two pairs of sets of paralogous protein sequences by means of the phylogenetic tree topology derived from the small subunit ribosomal RNA, even if this is rooted in all three possible ways. The thermophily index of all the reconstructed ancestral sequences of the LUCA belongs to the set of the thermophile/hyperthermophile sequences, thus supporting the hypotheses that see the LUCA as a thermophile or a hyperthermophile.     

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

Summary A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes. Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, April 3, 1986     

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: spatial propagation of differential flexibility in rubredoxin hybrids

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.     

Ribosomes, Polyribosomes, and Deoxyribonucleic Acid from Thermophilic, Mesophilic, and Psychrophilic Clostridia

Analysis of deoxyribonucleic acid (DNA) from four species of Clostridium, including two thermophiles, a mesophile, and a psychrophile, revealed no obvious relationship between growth temperature and DNA base composition. The melting temperatures (T(m)) of the DNA from the four species varied no more among the thermophilic, mesophilic, and psychrophilic species than among many related mesophilic species. Characterization of ribosomes from the clostridia by means of optical rotatory dispersion yielded similar spectra in common with other unrelated organisms. Only small differences were noted in the base composition of ribosomal ribonucleic acid (RNA) and in the amino acid composition of ribosomal proteins, including half-cystine content, as determined by cysteic acid analysis, and accessible sulfhydryl groups, as determined by titration with dithiobis (2-nitrobenzoic acid). Except for the two thermophiles, the ribosomal protein electrophoretic patterns were dissimilar. No unusual thermal stability was manifested in the T(m) values of thermophile ribosomal RNA. However, thermophile ribosome T(m) values (69 C) were higher than were mesophile and psychrophile T(m) values (64 C). Ribosomes from the four clostridial species were also examined in regard to the effect of heat on their functional integrity, measured by their activity in poly U-directed (14)C-phenylaline incorporation, and their gross physical integrity, measured by sucrose gradient analysis. The T(d, 5) values (temperature which produces 50% inactivation after 5 min) was found to be 70 and 72 C for the two thermophiles C. tartarivorum and C. thermosaccharolyticum, respectively; 57 C for a mesophile, C. pasteurianum; and 53 C for a psychrophile, Clostridium sp. strain 69. At 55 C, little effect was seen on the thermophile ribosomes, but the mesophile ribosomes lost 90% of their activity in 1 hr, and psychrophile ribosomes lost 100% of their activity within 10 min. According to sucrose gradient profiles, heating at 55 C results in dissociation of mesophile ribosomes and aggregation of psychrophile ribosomes. Thermophile S-100 fractions were also more thermostable than were mesophile or psychrophile S-100 fractions. The T(d, 5) values were 69 C for C. tartarivorum and C. thermosaccharolyticum S-100 and 41 C for C. pasteurianum and Clostridium sp. strain 69 S-100. The effect of heat on the endogenous incorporation of (14)C-valine by polysomes was also examined. In the case of thermophile polysomes, the extent of incorporation at 55 and 37 C was about equal. In the case of mesophile and psychrophile polysomes, the extent at 55 C was 44 and 39%, respectively, of the value at 37 C. The initial rates of incorporation in all four cases were greater at 55 C than at 37 C.     

Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae.

Protein unfolding of eight different phycocyanins was investigated utilizing circular dichroism and visible spectra. The phycocyanin samples were extracted from algae that are normally found in vastly different environments, and are classified as mesophilic, thermophilic, halophilic and psychrophilic. The ability of these proteins to resist the denaturant urea is in the order of thermophile greater than mesophile, halophile greater than psychrophile. Based on a two-state approximation the apparent free energies of protein unfolding at zero urea denaturant concentration, deltaGH2Oapp, were found to range from 2.4 to 8.8 kcal/mole for the eight phycocyanins at pH 6 and 25 degrees C. The proteins from the thermophile are generally more stable than those from the mesophile. An extra stability of the halophile is believed due to the specific interaction of the proteins and the ions in solution. A correction for deltaGH2Oapp due to minor amino acid differences reveals that the stability and the structural properties of these proteins are primarily affected by this minor difference in amino acid compositions.     

Thermophilicity of wild type and mutant cold shock proteins by molecular dynamics simulation

In the attempt to clarify possible mechanisms underlying thermal stability of proteins, we study through molecular dynamics thermophile Bc-Csp, mesophile Bs-CspB, and selected mutants. These proteins have been extensively characterized experimentally; researchers showed that differential thermostability among the wild type proteins is fundamentally linked to one or two mutated amino acids, and that the nature of the effect is electrostatic. They also inferred an atomistic mechanism related to removal of unfavorable interactions, rather than to the formation of salt bridges. Molecular dynamics allows us to confirm and support both hypotheses. Several other collective parameters have also been monitored in relation to thermophilicity, such as global and local rigidity, permanence and number of hydrogen bonds, or of salt links. None of these clearly correlates with the thermal stability of the presently studied proteins.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.     

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.     

The late stage of genetic code structuring took place at a high temperature

The correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter more frequently into (hyper)thermophile proteins is used to conduct an analysis aiming to establish whether genetic code structuring took place at a low or a high temperature. If the number of codons attributed to the various amino acids in the genetic code constitutes an estimate of the mean amino acid composition of proteins produced when the genetic code was definitively structured, then the thermophily index can also be associated to the genetic code. This value and the sampling of the variable thermophily index of different alignments of protein sequences from mesophile, thermophile and hyperthermophile species make it possible to establish, with an extremely high statistical confidence, that the late stage of genetic code structuring took place in a hyperthermophile (or thermophile) 'organism'. Moreover the 95% confidence interval of the temperature at which the genetic code was fixed turned out to be 91+/-24 degrees C. These observations seem to support the hypothesis that the origin of life might have taken place at a high temperature.     

Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein

The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential.     

Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.     

The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.