摘要
比较极端嗜热蛋白与同源嗜温蛋白,有显著性差别的是极端嗜热蛋白含有较多的盐桥及带电残基,其他参数的差异仅仅有统计倾向性。然而,大量有关电荷相互作用是否维持蛋白稳定的实验结果却相互矛盾。本工作以极端嗜热蛋白Ssh10b为模型,主要研究盐桥以及带电残基与蛋白热稳定性的内在联系。
理论分析提出:盐桥对温度变化的极度不敏感使得它成为蛋白在高温条件下维持稳定的重要因素。这一理论很好的解释了盐桥与蛋白热稳定性的内在关系,但是,至今没有实验证据。本研究通过双突变循环测定了Ssh10b分子中两个最为保守的盐桥在不同温度下的耦合自由能。结果显示,尽管Ssh10b的稳定自由能随着温度升高而显著下降,但这两个盐桥对蛋白稳定能的直接贡献却几乎不随温度改变,证明了盐桥是高温条件下维持蛋白稳定的重要因素。
通过比较本研究的实验结果和一些已经发表的数据,我们发现总体上那些由两个在一级序列上离得较远的残基所形成的盐桥对蛋白稳定能的直接贡献明显要大于那些由两个在一级序列上离得很近的残基所形成的盐桥,这一发现对蛋白质工程颇有意义。
本研究中,我们还通过理论分析提出:在中性pH区域,带电残基可解离基团的pKa漂移所引起的蛋白天然态构象熵增加可以促进蛋白稳定。酸碱滴定实验显示,相对于变性态,天然态Ssh10b中有许多可解离基团的pKa发生了漂移。理论计算结果显示一些碱性残基可解离基团的pKa发生了向下漂移,这使得在中性pH区域,天然态时构成蛋白的酸碱平衡分布远比在变性态中更为混乱,由此增加了天然态蛋白的构象熵,从而极大地提高了Ssh10b稳定性。
此外,为了研究脯氨酸残基对蛋白稳定的贡献,我们表达纯化了一系列有关脯氨酸的Ssh10b突变体,进行了相关的去折叠实验;提出了一个包含脯氨酸异构化平衡的去折叠模型,根据模型,脯氨酸残基对蛋白稳定能的贡献依赖于其天然态及变性态异构化平衡分布;实验结果与模型预测完全吻合。实验结果还显示,天然态时脯氨酸的异构化导致的构象熵增加可以促进Ssh10b的稳定,从而间接为可解离基团pKa漂移可以促进蛋白稳定这一观点提供了实验证据。
A statistical research showed that comparing the structural parameters between hyperthermophilic proteins and mesophilic ones, the only generally observed rule is an increase in the number of salt bridges and charged amino acid residues with increasing growth temperature. However, estimates of the energy contribution of electrostatic interaction to protein stability have led to conflicting conclusions. In this work, we took the hyperthermophilic protein Ssh10b as model, made some researches mainly focused on the relationships between enhanced protein stability and salt bridges/charged residues.
Theoretical research showed that salt bridges are extremely resilient to temperature increases and thus are specially suited to promoting protein stability at high temperatures. However, so far there are no reports of experiments to measure the stability contribution of salt bridges at high temperatures and to provide evidence for these theoretical predictions. In this work, a double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in protein Ssh10b. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures.
Moreover, comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the direct contribution to protein stability of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.
In this work, we also proposed through theoretical analysis that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability. Acid/base titration demonstrated that relative to the unfolded state, the pKa values of many ionizable residues are shifted in the native Ssh10b. Theoretical prediction suggested that the pKa values of some alkali residues are downshifted in the native state, which should result in that at neutral pH region, the native Ssh10b is existed as a conformation ensemble with these ionizable residues in more disordered acid/base equilibrium. Theoretical calculation indicated that the increased conformational entropy in native state induced by pKa shifts at neutral pH region could stabilize the protein greatly.
In addition, in order to study the stability contribution of the proline residues, in this work, we expressed and purified a series of Ssh10b mutants about proline, made correlative unfolding experiments in detail. We proposed a new unfolding mechanism including proline isomerization. According to this model, the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between the cis- and trans- isomers in both unfolded and folded states, agreeing well with unfolding experimental results. The results also demonstrated that the increased conformational entropy due to two native forms induced by proline isomerization contributes to stabilize the folded protein, providing experimental evidence indirectly for the viewpoint that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability.
引文
[1] Adams M W. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu Rev Microbiol, 1993, 47:627-658.
[2] Stetter K O. Extremophiles and their adaptation to hot environments. FEBS Lett, 1999, 452:22-25.
[3] Vieille C, Zeikus J G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev, 2001, 65:1-43.
[4] Vieille C, Burdette D S, Zeikus J G. Thermozymes. Biotechnol Annu Rev, 1996, 2:1-83.
[5] Zeikus J G, Vieille C, Savchenko A. Thermozymes: biotechnology and structure-function relationships. Extremophiles, 1998, 2:179-183.
[6] Vieille C, Hess J M, Kelly R M, Zeikus J G. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol, 1995, 61:1867-1875.
[7] Russell R J, Ferguson J M, Hough D W, et al. The crystal structure of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 A resolution. Biochemistry, 1997, 36:9983-9994.
[8] Bauer M W, Kelly R M. The family 1 beta-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry, 1998, 37:17170-17178.
[9] Stetter K O. Hyperthermophiles in the history of life. Ciba Found Symp, 1996, 202:1–10.
[10] Matthews B W, Weaver L H, Kester W H. The conformation of thermolysin. J Biol Chem, 1974, 249:8030-8044.
[11] Perutz M, Raidt H. Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2. Nature, 1975, 255:256-259.
[12] Fields P A. Protein function at thermal extremes: balancing stability and flexibility. Comp Biochem Physiol A Mol Integr Physiol, 2001, 129:417-431.
[13] Argos P, Rossmann M G, Grau U M, et al. Thermal stability and protein structure. Biochemistry, 1979, 18:5698-5703.
[14] Kn?chel T R, Hennig M, Merz A, et al. The crystal structure of indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus in three different crystal forms: effects of ionic strength. J Mol Biol, 1996, 262:502-515.
[15] Chakravarty S, Varadarajan R. Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study. Biochemistry, 2002, 41:8152-8161.
[16] Bohm G, Jaenicke R. Relevance of sequence statistics for the properties of extremophilic proteins. Int J Pept Protein Res, 1994, 43:97-106.
[17] Qyerol E, Perez-Pons J A, and Mozo-Villarias A. Analysis of protein conformational characteristics related to thermostability. Protein Eng, 1996, 9:256-271.
[18] Kumar S, Tsai C, Nussinov R. Factors enhancing protein thermostability. Protein Eng, 2000, 13:179-191.
[19] Woolfson D N, Williams D H. The influence of proline residues on alpha-helical structure. FEBS Lett, 1990, 277:185-188.
[20] Kumar S, Bansal M. Structural and sequence characteristics of long alpha helices in globular proteins. Biophys J, 1996, 71:1574-1586.
[21] Kumar S, Bansal M. Dissecting alpha-helices: position-specific analysis of alpha-helices in globular proteins. Proteins, 1998, 31:460-476.
[22] Kumar S, Bansal M. Geometrical and sequence characteristics of alpha-helices in globular proteins. Biophys J, 1998, 75:1935-1944.
[23] Auerbach G, Huber R, Gr?ttinger M, et al. Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability. Structure, 1997, 5:1475-1483.
[24] Nicholson H, Becktel W J, Matthews B W. Enhanced protein thermostability from designed mutations that interact with alpha-helix dipoles. Nature, 1988, 336:651-656.
[25] Brown S H, Kelly R M. Characterization of amylolytic enzymes, having both alpha-1,4 and alpha-1,6 hydrolytic activity, from the thermophilic archaea Pyrococcus furiosus and Thermococcus litoralis. Appl Environ Microbiol, 1993, 59:2614-2621.
[26] Karshikoff A, Ladenstein R. Proteins from thermophilic and mesophilic organisms essentially do not differ in packing. Protein Eng, 1998, 11:867-872.
[27] Adams M W, Kelly R M. Enzymes from microorganisms from extreme environments. C&E News, 1995, 73:32-42.
[28] Andreotti G, Cubellis M V, Nitti G, et al. An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta, 1995, 1247:90-96.
[29] Bonisch H, Backmann J, Kath T, et al. Adenylate kinase from Sulfolobus acidocaldarius: expression in Escherichia coli and characterization by Fourier transform infrared spectroscopy. Arch Biochem Biophys, 1996, 333:75-84.
[30] Vihinen M. Relationship of protein flexibility to thermostability. Protein Eng, 1987, 1:477-480.
[31] Gershenson A, Schauerte J A, Giver L, et al. Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry, 2000, 39:4658-4665.
[32] Jaenicke R, B?hm G. The stability of proteins in extreme environments. Curr Opin Struct Biol, 1998, 8:738-748.
[33] Manco G, GiosuèE, D'Auria S, et al. Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch Biochem Biophys, 2000, 373:182-192.
[34] Závodszky P, Kardos J, Svingor, et al. Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc Natl Acad Sci USA, 1998, 95:7406-7411.
[35] Beaucamp N, Hofmann A, Kellerer B, et al. Dissection of the gene of the bifunctional PGK-TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: design and characterization of the separate triosephosphate isomerase. Protein Sci, 1997, 6:2159-2165.
[36] D'Auria S, Nucci R, Rossi M, et al. Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols. J Biochem, 1999, 126:545-552.
[37] de Bakker P I, Hünenberger P H, McCammon J A. Molecular dynamics simulations of the hyperthermophilic protein Sac7d from Sulfolobus acidocaldarius: contribution of salt bridges to thermostability. J Mol Biol, 1999, 285:1811-1830.
[38] Kujo C, Oshima T. Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents. Appl Environ Microbiol, 1998, 64:2152-2157.
[39] Rüdiger A, Jorgensen P L, Antranikian G. Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli. Appl Environ Microbiol, 1995, 61:567-575.
[40] Sako Y, Croocker P C, Ishida Y. An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1. FEBS Lett, 1997, 415:329-334.
[41] Wilquet V, Gaspar J A, van de Lande M, et al. Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase. Eur J Biochem, 1998, 255:628-637.
[42] Ichikawa J K, Clarke S. A highly active protein repair enzyme from an extreme thermophile: the L-isoaspartyl methyltransferase from Thermotoga maritima. Arch Biochem Biophys, 1998, 358:222-231.
[43] Merz A, Kn?chel T, Jansonius J N, et al. The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritime is destabilized by mutational disruption of two solvent-exposed salt bridges. J Mol Biol, 1999, 288:753-763.
[44] Sterner R, Kleemann G R, Szadkowski H, et al. Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer. Protein Sci, 1996, 5:2000-2008.
[45] Hernandez G, Jenney Jr F E, Adams M W, et al. Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature. Proc Natl Acad Sci USA, 2000, 97:3166-3170.
[46] Fitter J, Heberle J. Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase. Biophys J, 2000, 79:1629-1636.
[47] Panasik N, Brenchley J E, Farber G K. Distributions of structural features contributing to thermostability in mesophilic and thermophilic alpha/beta barrel glycosyl hydrolases. Biochim Biophys Acta, 2000, 1543:189-201.
[48] Fitter J, Herrmann R, Dencher N A, et al. Activity and stability of a thermostable alpha- amylase compared to its mesophilic homologue: mechanisms of thermal adaptation. Biochemistry, 2000, 40:10723-10731.
[49] Lazaridis T, Lee I, Karplus M. Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin. Protein Sci, 1997, 6:2589-2605.
[50] Britton K L, Baker P J, Borges K M, et al. Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis. Eur J Biochem, 1995, 229:688-695.
[51] Zhu W, Sandman K, Lee G E, et al. NMR structure and comparison of the archaeal histone HFoB from the mesophile Methanobacterium formicicum with HMfB from the hyperthermophile Methanothermus fervidus. Biochemistry, 1998, 37:10573-10580.
[52] Li W, Grayling R A, Sandman K, et al. Thermodynamic stability of archaeal histones. Biochemistry, 1998, 37:10563-10572.
[53] Knapp S, Kardinahl S, Hellgren N, et al. Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 A resolution. J Mol Biol, 1999, 285:689-702.
[54] Grabarse W, Vaupel M, Vorholt J A, et al. The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri. Structure, 1999, 7:1257-1268.
[55] Lee Y E, Lowe S E, Henrissat B, et al. Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI. J Bacteriol, 1993, 175:5890-5898.
[56] Winterhalter C, Heinrich P, Candussio A, et al. Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol, 1995, 15:431-444.
[57] Thoma R, Hennig M, Sterner R, et al. Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima. Structure, 2000, 8:265-276.
[58] Kirino H, Aoki M, Aoshima M, et al. Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus. Eur J Biochem, 1994, 220:275-281.
[59] Moriyama H, Onodera K, Sakurai M, et al. The crystal structures of mutated 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 and their relationship to the thermostability of the enzyme. J Biochem, 1995, 117:408-413.
[60] Nesper M, Nock S, Sedlak E, et al. Dimers of Thermus thermophilus elongation factors Ts are required for its function as a nucleotide exchange factor of elongation factor Tu. Eur J Biochem, 1998, 255:81-86.
[61] Rahman R N, Fujiwara S, Nakamura H, et al. Ion pairs involved in maintaining a thermostable structure of glutamate dehydrogenase from a hyperthermophilic archaeon. Biochem Biophys Res Commun, 1998, 248:920-926.
[62] Shima S, Tziatzios C, Schubert D, et al. Lyotropic-salt-induced changes in monomer/ dimer/tetramer association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri in relation to the activity and thermostability of the enzyme. Eur J Biochem, 1998, 258:85-92.
[63] Vetriani C, Maeder D L, Tolliday N, et al. Protein thermostability above 100℃: a key role for ionic interactions. Proc Natl Acad Sci USA, 1998, 95:12300-12305.
[64] Hess D, Kruger K, Knappik A, et al. Dimeric 3-phosphoglycerate kinases from hyperthermophilic archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus. Eur J Biochem, 1995, 233:227-237.
[65] Voorhorst W G, Eggen R I, Luesink E J, et al. Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. J Bacteriol, 1995, 177:7105-7111.
[66] Chi Y I, Martinez-Cruz L A, Jancarik J, et al. Crystal structure of the beta-glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability. FEBS Lett, 1999, 445:375-383.
[67] Perutz M F. Electrostatic effects in proteins. Science, 1978, 201:1187-1191.
[68] Szilágyi A, Závodszky P. Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure, 2000, 8:493-504.
[69] Barril X, Aleman C, Orozco M, et al. Salt bridge interactions: stability of the ionic and neutral complexes in the gas phase, in solution, and in proteins. Proteins, 1998, 32:67-79.
[70] Dao-pin S, Anderson D E, Baase W A, et al. Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. Biochemistry, 1991, 30:11521-11529.
[71] Lebbink J H, Consalvi V, Chiaraluce R, et al. Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability. Biochemistry, 2002, 41:15524-15535.
[72] Lounnas V, Wade R C. Exceptionally stable salt bridges in cytochrome P450cam have functional roles. Biochemistry, 1997, 36:5402-5417.
[73] Pervushin K, Billeter M, Siegal G, et al. Structural role of a buried salt bridge in the 434 repressor DNA-binding domain. J Mol Biol, 1996, 264:1002-1012.
[74] Tissot A C, Vuilleumier S, Fersht A R. Importance of two buried salt bridges in the stability and folding pathway of barnase. Biochemistry, 1996, 35:6786-6794.
[75] Marti D N, Bosshard H R. Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges. J Mol Biol, 2003, 330:621-637.
[76] Marqusee S, Sauer R T. Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor. Protein Sci, 1994, 3:2217-2225.
[77] Serrano L, Horovitz A, Avron B, et al. Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. Biochemistry, 1990, 29:9343-9352.
[78] Singh U C. Probing the salt bridge in the dihydrofolate reductase-methotrexate complex by using the coordinate-coupled free-energy perturbation method. Proc Natl Acad Sci USA, 1988, 85:4280-4284.
[79] Sun D P, Sauer U, Nicholson H, et al. Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis. Biochemistry, 1991, 30:7142-7153.
[80] Yip K S, Stillman T J, Britton K L, et al. The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure, 1995, 3:1147-1158.
[81] Yip K S, Britton K L, Stillman T J, et al. Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family. Eur J Biochem, 1998, 255:336–346.
[82] Dougherty D A. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science, 1996, 271:163-168.
[83] Gallivan, J P, Dougherty D A. Cation-pi interactions in structural biology. Proc Natl Acad Sci USA, 1999, 96:9459-64.
[84] Singh J, Thornton J M. SIRIUS. An automated method for the analysis of the preferred packing arrangements between protein groups. J Mol Biol, 1990, 211:595-615.
[85] Flocco M M, Mowbray S L. Planar stacking interactions of arginine and aromatic side-chains in proteins. J Mol Biol, 1994, 235:709-717.
[86] Teplyakov A V, Kuranova I P, Harutyunyan E H, et al. Crystal structure of thermitase at 1.4 A resolution. J Mol Biol, 1990, 214:261-279.
[87] Ishikawa K, Okumura M, Katayanagi K, et al. Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution. J Mol Biol, 1993, 230:529-542.
[88] Serrano L, Bycroft M, Fersht A R. Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles. J Mol Biol, 1991, 218:465-475.
[89] Kannan N, Vishveshwara S. Aromatic clusters: a determinant of thermal stability of thermophilic proteins. Protein Eng, 2000, 13:753-761.
[90] Burley S K, Petsko G A. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science, 1985, 229:23-28.
[91] Georis J, de Lemos Esteves F, Brasseur, J, et al. An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Sci, 2000, 9:466-475.
[92] Li W F, Zhou X X, Lu P. Structural features of thermozymes. Biotechnol Adv, 2005, 23:271-281.
[93] Suvd D, Fujimoto Z, Takase K, et al. Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability. J Biochem, 2001, 129:461- 468.
[94] Tanner J J, Hecht R M, Krause K L. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 2.5 A resolution. Biochemist, 1996, 35:2597-2609.
[95] Tsai C J, Nussinov R. Hydrophobic folding units derived from dissimilar monomer structures and their interactions. Protein Sci, 1997, 6:24-42.
[96] Tsai C J, Nussinov R. Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association. Protein Sci, 1997, 6:1426-1437.
[97] Dill K A. Dominant forces in protein folding. Biochemestry, 1990, 31:7134-7155.
[98] Haney P, Konisky J, Koretke, K K, et al. Structural basis for thermostability and identification of potential active site residues for adenylate kinases from the archaeal genus Methanococcus. Proteins, 1997, 28:117-130.
[99] Salminen T, Teplyakov A, Kankare J, et al. An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases. Protein Sci, 1996, 5:1014-1025.
[100] Pace C N. Contribution of the hydrophobic effect to globular protein stability. J Mol Biol, 1992, 226:9-35.
[101] Ishikawa K, Nakamura H, Morikawa K, et al. Stabilization of Escherichia coli ribonuclease HI by cavityfilling mutations within a hydrophobic core. Biochemist, 1993, 32:6171-6178.
[102] Cacciapuoti G, Porcelli M, Bertoldo C, et al. Purification and characterization of extremely thermophilic and thermostable 5’-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds. J Biol Chem, 1994, 269:24762-24769.
[103] Pantoliano M W, Ladner R C, Bryan P N, et al. Protein engineering of subtilisin BPN’: enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry, 1987, 26:2077-2082.
[104] Takagi H, Takahashi T, Momose H, et al. Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease. J Biol Chem, 1990, 265:6874-6878.
[105] Mansfeld J, Vriend G, Dijkstra B W, et al. Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. J Biol Chem, 1997, 272:11152-11156.
[106] Nishikawa S, Adiwinata J, Morioka H, et al. A thermoresistant mutant of ribonuclease T1 having three disulfide bonds. Protein Eng, 1990, 3:443-448.
[107] Durrschmidt P, Mansfeld J, Ulbrich-Hofmann R. Differention between coformatioal and autoproteolytic stability of the neutral protease from Bacillus stearothermophilus containing an engineered disulfide bond. Eur J Biochem, 2001, 268:3612-3618.
[108] Matsumura M, Signor G, Matthews B W. Substantial increase of protein stability by multiple disulphide bonds. Nature, 1989, 342:291-293.
[109] Volkin D B, Klibanov A M. Thermal destruction processes in proteins involving cystine residues. J Biol Chem, 1987, 262:2945-2950.
[110] Choi I G, Bang W G, Kim S H, et al. Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization. J Biol Chem, 1999, 274:881-888.
[111] Kataeva I A, Uversky V N, Ljungdahl L G. Calcium and domain interactions contribute to the thermostability of domains of the multimodular cellobiohydrolase, CbhA, a subunit of the Clostridium thermocellum cellulosome. Biochem J, 2003, 372:151-161.
[112] Radfar R, Leaphart A, Brewer J M, et al. Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica. Biochemist, 2000, 39:14481-14486.
[113] Smith C A, Toogood H S, Baker H M, et al. Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution. J Mol Biol, 1999, 294:1027-1040.
[114] Kojoh K, Matsuzawa H, Wakagi T. Zinc and an N-terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature. Eur J Biochem, 1999, 264:85-91.
[115] Gr?ttinger M, Dankesreiter A, Schurig H, et al. Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: catalytic, spectral and thermodynamic properties. J Mol Biol, 1998, 280:525-533.
[116] Andr? S, Frey G, Jaenicke R, et al. The thermosome from Methanopyrus kandleri possesses an NH4+-dependent ATPase activity. Eur J Biochem, 1998, 255:93-99.
[117] Arnone M I, Birolo L, Pascarella S, et al. Stability of aspartate aminotransferase from Sulfolobus solfataricus. Protein Eng, 1997, 10:237-248.
[118] Ghosh M, Grunden A M, Dunn D M, et al. Characterization of native and recombinant forms of an unusual cobalt dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol, 1998, 180:4781-4789.
[119] Tomschy A, Glockshuber R, Jaenicke R. Functional expression of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli. Authenticity and kinetic properties of the recombinant enzyme. Eur J Biochem, 1993, 214:43-50.
[120] Cacciapuoti G, Fusco S, Caiazzo N, et al. Heterologous expression of 5’-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability. Protein Expr Purif, 1999, 16:125-135.
[121] Matussek K, Moritz P, Brunner N, et al. Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus. J Bacteriol, 1998, 180:5997-6004.
[122] Arnold U, Schierhorn A, Ulbrich-Hofmann R. Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation. Eur J Biochem, 1999, 259:470-475.
[123] Olsen O, Thomsen K K. Improvement of bacterial beta-glucanase thermostability by glycosylation. J Gen Microbiol, 1991, 137:579-585.
[124] Edmondson S P, Qui L, Shriver J W. Solution structure of the DNA-binding protein sac7d from the hyperthermophile Sulfolobus acidocaldarius. Biochemistry, 1995, 34:13289-13304.
[125] Maras B, Consalvi V, Chiaraluce R, et al. The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-epsilon-methyllysine related to thermostability? Eur J Biochem, 1992, 203:81-87.
[126] McAfee J G, Edmondson S P, Datta P K, et al. Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius. Biochemistry, 1995, 34:10063-10077.
[127] Ermler U, Merckel M C, Thauer R K, et al. Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri-new insights into salt-dependence and thermostability. Structure, 1997, 5:635-646.
[128] Fabry S, Hensel R. Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus. Eur J Biochem, 1987, 165:147-155.
[129] Breitung J, B?rner G, Scholz S, et al. Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran: tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri. Eur J Biochem, 1992, 210:971-981.
[130] Breitung J, Schmitz R A, Stetter K O, et al. N5, N10-methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/KM) and thermostability in the presence of salts. Arch Microbiol, 1991, 156:517-524.
[131] Klein A R, Breitung J, Linder D, et al. N5, N10-methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri. Arch Microbiol, 1993, 159:213-219.
[132] Ma K, Linder D, Stetter K O, et al. Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri. Arch Microbiol, 1991, 155:593-600.
[133] Ma K, Zirngibl C, Linder D, et al. N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri. Arch Microbiol, 1991, 156:43-48.
[134] Shima S, Hérault D A, Berkessel A, et al. Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanococcus kandleri. Arch Microbiol, 1998, 170:469-472.
[135] Hei D J, Clark D S. Pressure stabilization of proteins from extreme thermophiles. Appl Environ Microbiol, 1994, 60:932-939.
[136] Michels P C, Clark D S. Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen. Appl Environ Microbiol, 1997, 63:3985-3991.
[137] Miller J F, Nelson C M, Ludlow J M, et al. High pressure-temperature bioreactor: assays of thermostable hydrogenase with fiber optics. Biotechnol Bioeng, 1989, 34:1015-1021.
[138] Ladenstein R, Antranikian G. Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water. Adv Biochem Eng Biotechnol, 1998, 61:37-85.
[139] Choli T, Henning P, Wittmann-Liebold B, et al. Isolation, characterization, and microsequence analysis of a small basic methylated DNA binding protein from the archaebacterium Sulfolobus solfataricus. Biochim Biophys Acta, 1988, 950:193-203.
[140] Choli T, Wittmann-Liebold B, Reinhardt R. Microsequence analysis of DNA-binding proteins 7a, 7b, and 7e from the archaebacterium Sulfolobus acidocaldarius. J Biol Chem, 1988, 263:7087-7093.
[141] Dick J, Reinhardt R. The structure of DNA-binding proteins from eu- and archaebacteria. In C. O. Gualerzi and C. L. Pon (ed.), Bacterial chromatin. Springer-Verlag, New York, N Y, 1986:185-218.
[142] Grote M, Dijk J, Reinhardt R. Ribosomal and DNA binding proteins of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochim Biophys Acta, 1986, 873:405-413.
[143] Kimura M, Kimura J, Davie P, et al. The amino acid sequence of a small DNA binding protein from the archaebacterium Sulfolobus solfataricus. FEBS Lett, 1984, 176:176-178.
[144] Agback P, Baumann H, Knapp S, et al. Architecture of nonspecific protein-DNA interactions in the Sso7d-DNA complex. Nat Struct Biol, 1998, 5:579-584.
[145] Gao Y G, Su S Y, Robinson H, et al. The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA. Nat Struct Biol, 1998, 5:782-786.
[146] Robinson H, Gao Y G, McCrary B S, et al. The hyperthermophile chromosomal protein Sac7d sharply kinks DNA. Nature, 1998, 392:202-205.
[147] Grayling R A, Sandman K, Reeve J N. Archaeal DNA binding proteins and chromosome structure. Syst Appl Microbiol, 1994, 16:582-590.
[148] Baumann H, Knapp S, Lundback T, et al. Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus. Nat Struct Biol, 1994, 1:808-819.
[149] Lundback T, Hansson H, Knapp S, et al. Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus. J Mol Biol, 1998, 276:775-786.
[150] López-García P, Knapp S, Ladenstein R, et al. In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth. Nucleic Acids Res, 1998, 26:2322-2328.
[151] Forterre P, Confalonieri F, Knapp S. Identification of the gene encoding archeal-specific DNA-binding proteins of the Sac10b family. Mol Microbiol, 1999, 32:669-670.
[152] Xue H, Guo R, Wen Y, et al. An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion. J Bacteriol, 2000, 182:3929-3933.
[153] Bell S D, Botting C H, Wardleworth B N, et al. The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation. Science, 2002, 296:148-151.
[154] Marsh V L, Peak-Chew S Y, Bell S D. Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein Alba. J Biol Chem, 2005, 280:21122-21128.
[155] Wardleworth B N, Russell R J, Bell S D, et al. Structure of Alba: an archaeal chromatin protein modulated by acetylation. EMBO J, 2002, 21:4654-4662.
[156] Wang G, Guo R, Bartlam M, et al. Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii. Protein Sci, 2003, 12:2815-2822.
[157] Zhao K, Chai X, Marmorstein R. Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding. J Biol Chem, 2003, 278:26071-26077.
[158] Cui Q, Tong Y, Xue H, et al. Two conformations of archaeal Ssh10b. The origin of its temperature-dependent interaction with DNA. J Biol Chem, 2003, 278:51015-51022.
[159] Bohrmann B, Kellenberger E, Arnold-Schulz-Gahmen B, et al. Localization of histone-like proteins in thermophilic archaea by immunogold electron microscopy. J Struct Biol, 1994, 112:70-78.
[160] Guo R, Xue H, Huang L. Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo. Mol Microbiol, 2003, 50:1605-1615.
[161] Aravind L, Lakshminarayan M I, Anantharamana V. The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism. Genome Biology, 2003, 4:R64.
[162] Xu S, Qin S, Pan X M. Thermal and conformational stability of Ssh10b protein from archaeon Sulfolobus shibattae. Biochem J, 2004, 382:433-440.
[163] Barlow D J, Thornton, J M. Ion-pairs in proteins. J Mol Biol, 1983, 168:867-885.
[164] Mao Y J, Sheng X R, Pan X M. The effects of NaCl concentration and pH on the stability of hyperthermophilic protein Ssh10b. BMC Biochemistry, 2007, 8:28.
[165] Kelly S M, Jess T J, Price N C. How to study proteins by circular dichroism. Biochim Biophys Acta, 2005, 1751:119-139.
[166] Sreerama N, Woody R W. Computation and analysis of protein circular dichroism spectra. Methods Enzymol, 2004, 383:318-351.
[167] Rogers D M, Hirst J D. Calculations of protein circular dichroism from first principles. Chirality, 2004, 16:234-243.
[168] Morrisett J D, David J S, Pownall H J, et al. Interaction of an apolipoprotein (apoLP-alanine) with phosphatidylcholine. Biochemistry, 1973, 12:1290-1299.
[169] Chang C T, Wu C S, Yang J T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem, 1978, 91:13-31.
[170] Sreerama N, Woody R W. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem, 2000, 287:252-260.
[171] Hennessey Jr J P, Johnson Jr W C. Information content in the circular dichroism of proteins. Biochemstry, 1981, 20:1085-1094.
[172] Pancoska P, Bitto E, Janota V, et al. Comparison of and limits of accuracy for statistical analyses of vibrational and electronic circular dichroism spectra in terms of correlations to and predictions of protein secondary structure. Protein Sci, 1995, 4:1384-1401.
[173] Sreerama N, Venyaminov S Y, Woody R W. Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Protein Sci, 1999, 8:370-380.
[174] Sreerama N, Woody R W. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal Biochem, 1993, 209:32-44.
[175] Manavalan P, Johnson Jr W C. Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem, 1987, 167:76- 85.
[176] Johnson W C. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins, 1999, 35:307-312.
[177] Andrade M A, Chacón P, Merelo J J, et al. Evaluation of secondary structure of proteins from UV circular dichroism using an unsupervised neural network. Protein Eng, 1993, 6:383-390.
[178] Provencher S W, Gl?ckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry, 1981, 20:33-37.
[179] Greenfield N J. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal Biochem, 1996, 235:1-10.
[180] Lobley A, Whitmore L, Wallace B A. DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics, 2002, 18:211-212.
[181] Whitmore L, Wallace B A. DICHROWEB, An online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res, 2004, 32:W668-W673.
[182] Lees J G, Miles A J, Wien F, et al. A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics, 2006, 22:1955-1962.
[183] Lees J G, Miles A J, Janes R W, et al. Novel methods for secondary structure determination using low wavelength (VUV) circular dichroism spectroscopic data. BMC Bioinformatics, 2006, 7:507.
[184] Kelly S M, Price N C. The application of circular dichroism to studies of protein folding and unfolding. Biochim Biophys Acta, 1997, 1338:161-185.
[185] Freskg?rd P O, M?rtensson L G, Jonasson P, et al. Assignment of the contribution of the tryptophan residues to the circular dichroism spectrum of human carbonic anhydrase II. Biochemistry, 1994, 33:14281-14288.
[186] Woody A Y, Woody R W. Individual tyrosine side-chain contributions to circular dichroism of ribonuclease. Biopolymers, 2003, 72:500-513.
[187] Krell T, Horsburgh M J, Cooper A, et al. Localization of the active site of type II dehydroquinases. Identification of a common arginine-containing motif in the two classes of dehydroquinases. J Biol Chem, 1996, 271:24492-24497.
[188] Andersson L A, Peterson J A. Active-site analysis of ferric P450 enzymes: hydrogen-bonding effects on the circular dichroism spectra. Biochem Biophys Res Commun, 1995, 211:389-395.
[189] Alden R G, Johnson E, Nagarajan V, et al. Calculations of spectroscopic properties of the LH2 bacteriochlorophyll-protein antenna complex from Rhodopseudomonas acidophila. J Phys Chem B, 1997, 101:4667-4680.
[190] Missiakas D, Betton J M, Minard P, et al. Unfolding–refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains. Biochemistry, 1990, 29:8683-8689.
[191] Munro A W, Lindsay J G, Coggins J R, et al. Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3. FEBS Lett, 1994, 343:70-74.
[192] Boxer D H, Zhang H, Gourley D G, et al. Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE. Org Biomol Chem, 2004, 2:2829-2837.
[193] Anderson L A, Palmer T, Price N C, et al. Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli. Eur J Biochem, 1997, 246:119-126.
[194] Hope J, Shearman M S, Baxter H C, et al. Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer’s disease amyloid peptide, Ah 25-35. Neurodegeneration, 1996, 5:1-11.
[195] Pandya M J, Cerasoli E, Joseph A, et al. Sequence and structural duality: designing peptides to adopt two stable conformations. J Am Chem Soc, 2004, 126:17016-17024.
[196] Radford S E. Protein folding: progress made and promises ahead. Trends Biochem Sci, 2000, 25:611-618.
[197] Jemth P, Gianni S, Day R, et al. Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation. Proc Natl Acad Sci USA, 2004, 101:6450-6455.
[198] Dobson C M. Experimental investigation of protein folding and misfolding. Methods, 2004, 34:4-14.
[199] Woese C R, Kandler O, Wheelis M L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA, 1990, 87:4576-4579.
[200] Russell R J, Hough D W, Danson M J, et al. The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum. Structure, 1994, 2:1157-1167.
[201] Spassov V Z, Karshikoff A D, Ladenstein R. The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions. Protein Sci, 1995, 4:1516-1527.
[202] Schumann J, Bohm G, Schumacher G, et al. Stabilization of creatinase from Pseudomonas putida by random mutagenesis. Protein Sci, 1993, 10:1612-1620.
[203] Vieille C, Zeikus J G. Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol, 1996, 14:183-191.
[204] Hennig M, Darimont B, Sterner R, et al. 2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability. Structure, 1995, 3:1295-1306.
[205] Jaenicke R, Schurig H, Beaucamp N, et al. Structure and stability of hyperstable proteins: glycolytic enzymes from hyperthermophilic bacterium Thermotoga maritima. Advan Protein Chem, 1996, 48:181-269.
[206] Korndorfer I, Steipe B, Huber R, et al. The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima at 2.5 A resolution. J Mol Biol, 1995, 246:511-521.
[207] Kawamura S, Tanaka I, Yamasaki N, et al. Contribution of a salt bridge to the thermostability of DNA binding protein HU from Bacillus stearothermophilus determined by site-directed mutagenesis. J Biochem, 1997, 121:448-455.
[208] Waldburger C D, Schildbach J F, Sauer R T. Are buried salt bridges important for protein stability and conformational specificity? Nature Struct Biol, 1995, 2:122-128.
[209] Hendsch Z S, Tidor B. Do salt bridges stabilize proteins? A continuum electrostatic analysis. Protein Sci, 1994, 3:211-226.
[210] Elcock A H. The stability of salt bridges at high temperatures: implications for hyperthermophilic proteins. J Mol Biol, 1998, 284:489-502.
[211] Thomas A S, Elcock A H. Molecular simulations suggest protein salt bridges are uniquely suited to life at high temperatures. J Am Chem Soc, 2004, 126:2208-2214.
[212] Krylov D, Barchi J, Vinson C. Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids. J Mol Biol, 1998, 279:959-972.
[213] Vaughan C K, Harryson P, Buckle A M, et al. A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase. Acta Crystallogr D Biol Crystallogr, 2002, 58:591-600.
[214] Luisi D L, Snow C D, Lin J J, et al. Surface salt bridges, double-mutant cycles, and protein stability: an experimental and computational analysis of the interaction of the Asp 23 side chain with the N-terminus of the N-terminal domain of the ribosomal protein L9. Biochemistry, 2003, 42:7050-7060.
[215] Makhatadze G I, Loladze V V, Ermolenko D N, et al. Contribution of surface salt bridges to protein stability: guidelines for protein engineering. J Mol Biol, 2003, 327:1135-1148.
[216] Neet K E, Timm D E. Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. Protein Sci, 1994, 3:2167-2174.
[217] Schellman J A. The thermodynamic stability of proteins. Annu Rev Biophys Chem, 1987, 16:115-137.
[218] Chen B L, Schellman J A. Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies. Biochemistry, 1989, 28:685-691.
[219] Agashe V R, Udgaonkar J B. Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. Biochemistry, 1995, 34:3286-3299.
[220] Kamal J K, Nazeerunnisa M, Behere D V. Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters. J Biol Chem, 2002, 277:40717-40721.
[221] Santoro M M, Bolen D W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulphonyl alpha-chymotrypsin using different denaturants. Biochemistry, 1988, 27:8063-8068.
[222] Freire E. Thermal denaturation methods in the study of protein folding. Methods Enzymol, 1995, 259:144-168.
[223] Bosshard H R, Marti D N, Jelesarov I. Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings. J Mol Recognit, 2004, 17:1-16.
[224] Nojima H, Ikai A, Oshima T, et al. Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change. J Mol Biol, 1977, 116:429-442.
[225] Nojima H, Ikai A, Noda H, et al. Thermodynamic studies on reversible denaturation of thermostable proteins from an extreme thermophile. In Biochemistry of Thermophily (Friedman, S. M. Ed.), Academic Press, New York, 1978:305-323.
[226] Horovitz A, Serrano L, Avron B, et al. Strength and co-operativity of contributions of surface salt bridges to protein stability. J Mol Biol, 1990, 216:1031-1044.
[227] Sali D, Bycroft M, Fersht A R. Surface electrostatic interactions contribute little of stability of barnase. J Mol Biol, 1991, 220:779-788.
[228] Strop P, Mayo S L. Contribution of surface salt bridges to protein stability. Biochemistry, 2000, 39:1251-1255.
[229] Blasie C A, Berg J M. Electrostatic interactions across a beta-sheet. Biochemistry, 1997, 36:6218-6222.
[230] Ibarra-Molero B, Zitzewitz J A, Matthews C R. Salt-bridges can stabilize but do not accelerate the folding of the homodimeric coiled-coil peptide GCN4-p1. J Mol Biol, 2004, 336:989-996.
[231] Merkel J S, Sturtevant J M, Regan L. Side chain interactions in parallel beta sheets: the energetics of cross-strand pairings. Structure, 1999, 7:1333-1343.
[232] Lassila K S, Datta D, Mayo S L. Evaluation of the energetic contribution of an ionic network to beta-sheet stability. Protein Sci, 2002, 11:688-690.
[233] Nicholson H, Anderson D E, Dao-pin S, et al. Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme. Biochemistry, 1991, 30:9816-9828.
[234] Hong H, Szabo G, Tamm L K. Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening. Nat Chem Biol, 2006, 2:627-635.
[235] Kumar S, Nussinov R. How do thermophilic proteins deal with heat? Cell Mol Life Sci, 2001, 58:1216-1233.
[236] Elcock A H. Realistic modeling of the denatured states of proteins allows accurate calculations of the pH dependence of protein stability. J Mol Biol, 1999, 294:1051-1062.
[237] He Y, Xu J, Pan X M. A statistical approach to the prediction of pK(a) values in proteins. Proteins, 2007, 69:75-82.
[238] Huang Y W, Bolen D W. Covalent bond changes as a driving force in enzyme catalysis. Biochemistry, 1993, 32:9329-9339.
[239] Yao M, Bolen D W. How valid are denaturant-induced unfolding free energy measurements? Level of conformance to common assumptions over an extended range of ribonuclease A stability. Biochemistry, 1995, 34:3771-3781.
[240] Wisz M S, Hellinga H W. An empirical model for electrostatic interactions in proteins incorporating multiple geometry-dependent dielectric constants. Proteins, 2003, 51:360-377.
[241] Pickett S D, Sternberg M J. Empirical scale of side-chain conformational entropy in protein folding. J Mol Biol, 1993, 231:825-839.
[242] Suzuki Y, Oishi K, Nakano H, et al. A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1, 6-glucosidase. Appl Microbiol Biotechnol, 1987, 26:546-551.
[243] Suzuki Y. A general principle of increasing protein thermostability. Proc Jpn Acad B Phys Biol Sci, 1989, 65:146-148.
[244] Matthews B W, Nicholson H, Becktel W J. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA, 1987, 84:6663-6667.
[245] Hardy F, Veriend G, Veltman O R, et al. Stabilization of Bacillus stearotheromphilus neutral protease by introduction of prolines. FEBS Lett, 1993, 317:89-92.
[246] Watnabe K, Masada T, Ohashi H, et al. Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1, 6-glucosidase. Eur J Biochem, 1994, 226:277-283.
[247] Li Y, Reilly P J, Ford C. Effect of introducing proline residues on the stability of Aspergillus awamori. Protein Eng, 1997, 10:1199-1204.
[248] Allen M J, Countinho P M, Ford C F. Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations. Protein Eng, 1998, 11:783-788.
[249] Gaseidnes S, Synstad B, Jia X, et al. Stabilization of a chitinase from Serratia marcescens by Gly→Ala and Xxx→Pro mutations. Protein Eng, 2003, 16:841-846.
[250] Goihberg E, Dym O, Tel-Or, et al. A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase. Proteins, 2007, 66:196-204.
[251] Sriprapundh D, Vieille C, Zeikus J G. Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. Protein Eng, 2000, 13:259-265.
[252] Frare E, Laureto P, Scaramella E, et al. Chemical synthesis of the RGD-protein decorsin: Pro→Ala replacement reduces protein thermostability. Protein Eng Des Sel, 2005, 18:487-495.
[253] Saksguchi M, Matsuzaki M, Niimiya K, et al. Role of proline residues in conferring thermostability on aqualysin I. J Biochem, 2007, 142:213-220.
[254] Cheng, H N, Bovey F A. Cis-trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline. Biopolymers, 1977, 16:1465-1472.
[255] Grathwahl C, Wüthrich K. NMR studies of the rates of proline cis-trans isomerization in oligopeptides. Biopolymers, 1981, 20:2623-2633.
[256] Chien, C H, Huang L H, Chou C Y, et al. One site mutation disrupts dimer formation in human DPP-IV proteins. J Biol Chem, 2004, 279:52338-52345.
[257] Némethy, G, Leach, S J, Scheraga, H A. The influence of amino acid side chains on the free energy of helix-coil transitions. J Phys Chem, 1966, 70:998-1004.
[258] Nicholson H, Tronrud D E, Becktel W J, et al. Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme. Biopolymers, 1992, 32:1431-1441.