- Research article
- Open Access
The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog
© Cuneo et al; licensee BioMed Central Ltd. 2008
- Received: 08 November 2007
- Accepted: 28 March 2008
- Published: 28 March 2008
Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability.
We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 Å resolution. We find that tteRBP is significantly more stable ( app T m value ~102°C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) ( app T m value ~56°C). The tteRBP has essentially the identical backbone conformation (0.41 Å RMSD of 235/271 Cα positions and 0.65 Å RMSD of 270/271 Cα positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities.
The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.
- Amino Acid Diversity
- Immobilize Metal Affinity Chromatography
- Polar Amino Acid
- Circular Dichroism Signal
The mechanisms that contribute to protein thermal stability are varied, subtle, and complex [1–5]. Various contributing factors to thermal stability have been proposed by comparative analysis of thermophilic and mesophilic proteins [4, 6]. Proposed mechanisms can be categorized  generally as contributions by the main-chain structure (new folds , loop shortening ), or by side-chain interactions (increased packing in core  or surface , alteration of amino acid composition [11–13]), post-translational modifications  or co-factor binding [4, 15]). Usually increased stability arises from a combination of sequence- and structure-based adaptations resulting in a collection of improvements in the thermophilic protein compared to its mesophilic counterpart [4, 6, 16, 17]. Consequently, the determination of rules for thermal adaptations are difficult to dissect . Of particular interest, therefore, are pairs of naturally evolved proteins that are structurally very similar but differ substantially in thermal stability. Such pairs allow for the dissection of contributions by amino acid diversity to thermal stability in the absence of structural heterogeneity [17–20]. The structure of the Thermoanaerobacter tengcongensis ribose-binding (tteRBP) presented here reveals that this protein and its counterpart in the mesophilic Escherichia coli (ecRBP) form such a pair.
The ribose-binding proteins are members of the periplasmic binding protein (PBP) superfamily whose members play roles in prokaryotic ABC transport , chemotaxis [22, 23], and intercellular communication  systems. The PBP fold consists of two domains each of which adopts a three-layered α/β/α sandwich motif . The two domains are linked by two or three β-strands that form a flexible hinge which permits the domains of the protein to bend towards each other in response to ligand binding at the interface between the two domains [26–28].
Here we report the high-resolution X-ray crystallographic structure of a ribose binding protein (tteRBP) from the hyperthermophilic bacterium T. tengcongensis (optimal growth temp ~80°C) . We find that tteRBP has high sequence and structural similarity to the mesophilic E. coli RBP (ecRBP), although they differ markedly in their thermal stability. The near identity of backbone structure offers an opportunity to address local encoding of thermal stability by amino acid substitutions.
Thermal Stability and Ligand Binding
Binding of ribose to tteRBP was confirmed by observing ligand-mediated changes in the app T m value in the presence of 4.0 M GdCl. Under these conditions in the absence of ribose, the app T m value is 74°C; and 92°C in the presence of 1 mM ribose (Figure 2). The app T m value of the ribose complex in the absence of GdCl is 114°C; the app T m value for ecRBP under equivalent conditions is 72°C (Figure 2).
Overall Structure of tteRBP
Data collection and refinement statistics
Num. of Reflections (working set/test set)
Number of atoms
Bond lengths (Å)
Bond angles (°)
Average B-factor (Å 2 )
Ramachandran outliers (%)
Ramachandran favored (%)
Rotamer outliers (%)
Structural Diversity of tteRBP and ecRBP
Changes in the χ1 values of ecRBP and tteRBP. Non-conservative substitutions (as defined in ; charge inversions are scored as non-conservative here) are underlined.
Hydrogen bonding interactions in tteRBP and ecRBP
Side chain/Side chain
Side chain/Main chain
Main chain/Main chain
Amino Acid Diversity Among tteRBP and ecRBP
Divergence patterns in tteRBP and ecRBP. Classification of amino acids into core, boundary, or surface  allows identification of the regions which are conserved among tteRBP and ecRBP.
Amino acid sequence divergence as a function of core, boundary or surface in tteRBP and ecRBP. Differences are classified as substitutions which are found in tteRBP relative to ecRBP.
Non-Branched to Branched
Branched to Non-Branched
V to I
I to V
We have cloned, expressed, purified, and characterized the structure and stability of the ribose binding protein from the extremophilic bacterium T. tengcongensis. tteRBP is considerably more stable than ecRBP (46°C difference in app T m values of the apo proteins). The amino acid backbone structure of these two proteins are essentially identical (0.41 Å RMSD of 235/271 Cα positions and 0.65 Å RMSD of 270/271 Cα positions), suggesting that all the interactions contributing to differences in thermal stability are encoded entirely in the identity, location, and conformation of the amino acid side-chains.
Comparison of mesophilic and thermophilic protein structures has identified many structural adaptations which are postulated to confer thermal stability [2, 6, 11, 16–18, 38]. Numerous side-chain dependent contributions to thermal stability have been proposed, based on amino acid composition of thermophilic proteins and comparison of mesophilic and thermophilic protein sequences and structures, including; increased number of salt-bridges , differences in polar/apolar exposed and buried surface areas [8, 12, 39], introduction of prolines , introduction of disulfide bridges [41, 42], aromatic interactions , helix dipole stabilization , post-translational modification , alteration of amino acid packing [9, 10, 44] and secondary structure propensity of amino acids [8, 45].
The high structural similarity of the tteRBP/ecRBP pair allows for the dissection of amino acid diversity contributions to thermal stability in the absence of structural heterogeneity. The comparative analysis presented here shows that the substitutions responsible for conferring thermal stability on tteRBP are encoded in side-chain identity and location (core, boundary or surface) which serves to alter surface polarity/charge, removal of unsatisfied core hydrogen bonds and increase in core/boundary side-chain hydrophobicity. In the core of tteRBP there is a bias for the loss of polar amino acids and for the introduction of valine to isoleucine mutations which possibly lower the entropic contribution to the free energy of folding and limits burying core amino acids whose hydrogen bonding potential may remain unsatisfied [38, 46]. The large number of valine to isoleucine substitutions in the tteRBP core and boundary leads to an increase in side-chain hydrophobicity and increased packing [44, 47]. It is additionally observed in the boundary the substitution of non-β-branched amino acids for β-branched residues which has also been postulated to be important in increasing the packing . Additionally, in a trend that is also observed in other thermophilic proteins, the surface of tteRBP is generally more polar and charged with the introduction of an additional three polar residues and eleven charged residues.
The acquisition of thermal stability in tteRBP arises from contributions by side-chain mediated effects alone. This pair of proteins therefore provides a good test case to examine such contributions experimentally and address some long-standing questions in the acquisition of protein stability [1, 5, 49]: where in sequence and structure is stability encoded; how many mutations are needed; are mutations punctuated (single mutants cause large changes) or gradual, independent or correlated? Recent advances in protein fabrication automation  will assist in addressing these questions by enabling rapid construction of the many sequence variants needed.
Cloning Over-expression and Purification
The tte0206 gene was amplified from T. tengcongensis genomic DNA by the sticky-end PCR method using the following primers: PO4-TATGA AAACTATAGG ATTAGTGATATCTACTCTTAACAATCC, and TATGAAAACTATAGG ATTAGTGATATCTACTCTTAACAATCC for the 5' end of the gene; PO4- AATTCTAATGGTGATGGTGATGGTGTGATCCCTGTACATTTTCTTTTGTTATGAGTTTAAGTTCTGC, and CTAATGGTGATGGTGATGGTGTGATCCCTGTACATTTTCTTTTGTTATGAGTTTAAGTTCTGC for the 3' end of the gene . The resulting fragment was cloned into the Nde I/Eco RI sites of a pET21a (Novagen) plasmid for over-expression in E. coli. This ORF lacks the putative periplasmic signal sequence . The coding sequence starting at lysine 40 was cloned in-frame with an ATG start codon. A hexahistidine affinity tag and a glycine-serine linker was fused in-frame at the carboxy terminus to facilitate purification by immobilized metal affinity chromatography (IMAC). Protein concentration was determined spectrophotometrically (ε280 = 3800 M-1cm-1) . The resulting gene product was expressed and purified by IMAC as described . Pooled IMAC fractions were concentrated to 12 mL and were loaded onto a Superdex 26/60 S75 (Amersham) gel filtration column that was previously calibrated with blue dextran, bovine serum albumin, chicken serum albumin, chymotrypsin and lysozyme. tteRBP eluted from the column beginning at the void volume and ending at a calculated hydrodynamic radius corresponding to ~20 KDa. For crystallization and characterization, 10 mL fractions corresponding to a calculated hydrodynamic radius corresponding to an apparent molecular weight of 30 KDa ± 15 kDa, were collected and concentrated to 0.5 mM and dialyzed in 10 mM Tris pH7.8, 20 mM NaCl. An average of 30 mg of pure protein produced per liter of medium.
Circular dichroism (CD) measurements were determined on an Aviv Model 202 circular dichroism spectrophotometer. Thermal denaturations were determined by measuring the CD signal at 222 nm (1 cm path length) as a function of temperature, using 1 μM protein (10 mM Tris-HCl pH7.8, 150 mM NaCl), GdCl at various concentrations, in the presence or absence of 1 mM ribose. Protein samples were incubated for 15 minutes prior to collecting data. Each measurement includes a 3-second averaging time for data collection and a 60 second equilibration period at each temperature. Data was fit to a two-state model which accounts for the native and denatured baseline slopes, to determine the apparent T m values [31, 32]. It is not known whether equilibrium was achieved under these conditions; denaturation midpoint temperatures are therefore reported as apparent values ( app T m ). The app T m values in the absence of denaturant were determined by linear extrapolation .
Crystallization and Data Collection
Ribose was added to tteRBP in 3-fold stoichiometric excess prior to crystallization. tteRBP crystals were grown by micro-batch under paraffin oil in drops that contained 2 μl of the protein solution (0.5 mM) mixed with 2 μl of 0.1 M sodium citrate pH 4.0, 50% (w/v) PEG 1000 and 0.1 M potassium phosphate monobasic. The tteRBP crystals diffract to 1.9 Å resolution, belong to the C2 space group (a = 123.18 Å, b = 35.8 Å, c = 118.03 Å, β = 107.02) and typically grew within three weeks at 17°C (Table 1). No stabilizing cryoprotectant was used and crystals were frozen directly in precipitant solution, mounted in a nylon loop and flash frozen in liquid nitrogen. All data were collected at 100 K at the SER-CAT 22 BM beam line at the Advanced Photon Source. The diffraction data were scaled and indexed using SCALA and XDS [53, 54].
Structure Determination Methods, Model Building and Refinement
The tteRBP structure was determined by molecular replacement using the ribose-bound form of the ribose binding protein from E. coli  as the search model . Rotation, translation, and fitting functions revealed a clear solution yielding higher correlation coefficients and a lower R factor than all the others. Manual model building was carried out in the programs O and COOT and refined using REFMAC5 [55–57]. The final model for the tteRBP complex includes two intact tteRBP monomers (residues 2–275), two ribose molecules, and 346 water molecules. The model exhibits good stereochemistry as determined by PROCHECK and MolProbity; final refinement statistics are listed in Table 1[58, 59]. PDB coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the accession code 2IOY.
This study was funded grant by a grant from HSARPA (W81XWH-05-C-0161) to HWH, a Pioneer Award from the NIH (5 DP1 OD000122-02) to HWH, and a NIH sponsored Biological Chemistry training grant to MJC. The authors would like to acknowledge G. Shirman for protein expression and purification. Data were collected at the Southeast Regional Collaborative Access Team 22-BM at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.
- Vogt G, Woell S, Argos P: Protein thermal stability, hydrogen bonds, and ion pairs. J Mol Biol 1997, 269(4):631–643. 10.1006/jmbi.1997.1042View ArticleGoogle Scholar
- Kumar S, Tsai CJ, Nussinov R: Factors enhancing protein thermostability. Protein Eng 2000, 13(3):179–191. 10.1093/protein/13.3.179View ArticleGoogle Scholar
- Jaenicke R, Bohm G: The stability of proteins in extreme environments. Curr Opin Struct Biol 1998, 8(6):738–748. 10.1016/S0959-440X(98)80094-8View ArticleGoogle Scholar
- Vieille C, Zeikus GJ: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 2001, 65(1):1–43. 10.1128/MMBR.65.1.1-43.2001View ArticleGoogle Scholar
- Berezovsky IN, Shakhnovich EI: Physics and evolution of thermophilic adaptation. Proc Natl Acad Sci U S A 2005, 102(36):12742–12747. 10.1073/pnas.0503890102View ArticleGoogle Scholar
- Szilagyi A, Zavodszky P: Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 2000, 8(5):493–504. 10.1016/S0969-2126(00)00133-7View ArticleGoogle Scholar
- Kisker C, Schindelin H, Alber BE, Ferry JG, Rees DC: A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila. Embo J 1996, 15(10):2323–2330.Google Scholar
- Chakravarty S, Varadarajan R: Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study. Biochemistry 2002, 41(25):8152–8161. 10.1021/bi025523tView ArticleGoogle Scholar
- Goldstein RA: Amino-acid interactions in psychrophiles, mesophiles, thermophiles, and hyperthermophiles: insights from the quasi-chemical approximation. Protein Sci 2007, 16(9):1887–1895. 10.1110/ps.072947007View ArticleGoogle Scholar
- Glyakina AV, Garbuzynskiy SO, Lobanov MY, Galzitskaya OV: Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms. Bioinformatics 2007, 23(17):2231–2238. 10.1093/bioinformatics/btm345View ArticleGoogle Scholar
- Liang HK, Huang CM, Ko MT, Hwang JK: Amino acid coupling patterns in thermophilic proteins. Proteins 2005, 59(1):58–63. 10.1002/prot.20386View ArticleGoogle Scholar
- Fukuchi S, Nishikawa K: Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria. J Mol Biol 2001, 309(4):835–843. 10.1006/jmbi.2001.4718View ArticleGoogle Scholar
- Bohm G, Jaenicke R: Relevance of sequence statistics for the properties of extremophilic proteins. Int J Pept Protein Res 1994, 43(1):97–106.View ArticleGoogle Scholar
- Jafari-Aghdam J, Khajeh K, Ranjbar B, Nemat-Gorgani M: Deglycosylation of glucoamylase from Aspergillus niger: effects on structure, activity and stability. Biochim Biophys Acta 2005, 1750(1):61–68.View ArticleGoogle Scholar
- Fujii T, Hata Y, Oozeki M, Moriyama H, Wakagi T, Tanaka N, Oshima T: The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7. Biochemistry 1997, 36(6):1505–1513. 10.1021/bi961966jView ArticleGoogle Scholar
- Yano JK, Poulos TL: New understandings of thermostable and peizostable enzymes. Curr Opin Biotechnol 2003, 14(4):360–365. 10.1016/S0958-1669(03)00075-2View ArticleGoogle Scholar
- Razvi A, Scholtz JM: Lessons in stability from thermophilic proteins. Protein Sci 2006, 15(7):1569–1578. 10.1110/ps.062130306View ArticleGoogle Scholar
- Maes D, Zeelen JP, Thanki N, Beaucamp N, Alvarez M, Thi MH, Backmann J, Martial JA, Wyns L, Jaenicke R, Wierenga RK: The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures. Proteins 1999, 37(3):441–453. 10.1002/(SICI)1097-0134(19991115)37:3<441::AID-PROT11>3.0.CO;2-7View ArticleGoogle Scholar
- Kannan N, Vishveshwara S: Aromatic clusters: a determinant of thermal stability of thermophilic proteins. Protein Eng 2000, 13(11):753–761. 10.1093/protein/13.11.753View ArticleGoogle Scholar
- Greaves RB, Warwicker J: Mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles. BMC Struct Biol 2007, 7: 18. 10.1186/1472-6807-7-18View ArticleGoogle Scholar
- Boos W, Shuman H: Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 1998, 62(1):204–229.Google Scholar
- Davidson AL, Shuman HA, Nikaido H: Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A 1992, 89(6):2360–2364. 10.1073/pnas.89.6.2360View ArticleGoogle Scholar
- Bjorkman AJ, Binnie RA, Zhang H, Cole LB, Hermodson MA, Mowbray SL: Probing protein-protein interactions. The ribose-binding protein in bacterial transport and chemotaxis. J Biol Chem 1994, 269(48):30206–30211.Google Scholar
- Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM: Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell 2006, 126(6):1095–1108. 10.1016/j.cell.2006.07.032View ArticleGoogle Scholar
- Quiocho FA, Ledvina PS: Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol Microbiol 1996, 20(1):17–25. 10.1111/j.1365-2958.1996.tb02484.xView ArticleGoogle Scholar
- Bjorkman AJ, Mowbray SL: Multiple open forms of ribose-binding protein trace the path of its conformational change. J Mol Biol 1998, 279(3):651–664. 10.1006/jmbi.1998.1785View ArticleGoogle Scholar
- Magnusson U, Chaudhuri BN, Ko J, Park C, Jones TA, Mowbray SL: Hinge-bending motion of D-allose-binding protein from Escherichia coli: three open conformations. J Biol Chem 2002, 277(16):14077–14084. 10.1074/jbc.M200514200View ArticleGoogle Scholar
- Sharff AJ, Rodseth LE, Spurlino JC, Quiocho FA: Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. Biochemistry 1992, 31(44):10657–10663. 10.1021/bi00159a003View ArticleGoogle Scholar
- Bao Q, Tian Y, Li W, Xu Z, Xuan Z, Hu S, Dong W, Yang J, Chen Y, Xue Y, Xu Y, Lai X, Huang L, Dong X, Ma Y, Ling L, Tan H, Chen R, Wang J, Yu J, Yang H: A complete sequence of the T. tengcongensis genome. Genome Res 2002, 12(5):689–700. 10.1101/gr.219302View ArticleGoogle Scholar
- Nakai K, Horton P: PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 1999, 24(1):34–36. 10.1016/S0968-0004(98)01336-XView ArticleGoogle Scholar
- Schellman JA: The thermodynamic stability of proteins. Annu Rev Biophys Biophys Chem 1987, 16: 115–137. 10.1146/annurev.bb.16.060187.000555View ArticleGoogle Scholar
- Cohen DS, Pielak GJ: Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature. Protein Sci 1994, 3(8):1253–1260.View ArticleGoogle Scholar
- Cuneo MJ, Changela A, Warren JJ, Beese LS, Hellinga HW: The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites. J Mol Biol 2006, 362(2):259–270. 10.1016/j.jmb.2006.06.084View ArticleGoogle Scholar
- Navaza J: AMoRe: an automated package for molecular replacement. Acta Cryst 1994, A50: 157–163.View ArticleGoogle Scholar
- McDonald IK, Thornton JM: Satisfying hydrogen bonding potential in proteins. J Mol Biol 1994, 238(5):777–793. 10.1006/jmbi.1994.1334View ArticleGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22(22):4673–4680. 10.1093/nar/22.22.4673View ArticleGoogle Scholar
- Wisz MS, Hellinga HW: An empirical model for electrostatic interactions in proteins incorporating multiple geometry-dependent dielectric constants. Proteins 2003, 51(3):360–377. 10.1002/prot.10332View ArticleGoogle Scholar
- Weers PM, Abdullahi WE, Cabrera JM, Hsu TC: Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31. Biochemistry 2005, 44(24):8810–8816. 10.1021/bi050502vView ArticleGoogle Scholar
- Criswell AR, Bae E, Stec B, Konisky J, Phillips GN Jr.: Structures of thermophilic and mesophilic adenylate kinases from the genus Methanococcus. J Mol Biol 2003, 330(5):1087–1099. 10.1016/S0022-2836(03)00655-7View ArticleGoogle Scholar
- Watanabe K, Masuda T, Ohashi H, Mihara H, Suzuki Y: Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule. Eur J Biochem 1994, 226(2):277–283. 10.1111/j.1432-1033.1994.tb20051.xView ArticleGoogle Scholar
- Bian Y, Liang X, Fang N, Tang XF, Tang B, Shen P, Peng Z: The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease. FEBS Lett 2006, 580(25):6007–6014. 10.1016/j.febslet.2006.09.068View ArticleGoogle Scholar
- Takagi H, Takahashi T, Momose H, Inouye M, Maeda Y, Matsuzawa H, Ohta T: 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(12):6874–6878.Google Scholar
- Hennig M, Darimont B, Sterner R, Kirschner K, Jansonius JN: 2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability. Structure 1995, 3(12):1295–1306. 10.1016/S0969-2126(01)00267-2View ArticleGoogle Scholar
- Britton KL, Baker PJ, Borges KM, Engel PC, Pasquo A, Rice DW, Robb FT, Scandurra R, Stillman TJ, Yip KS: Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis. Eur J Biochem 1995, 229(3):688–695. 10.1111/j.1432-1033.1995.tb20515.xView ArticleGoogle Scholar
- Corazza A, Rosano C, Pagano K, Alverdi V, Esposito G, Capanni C, Bemporad F, Plakoutsi G, Stefani M, Chiti F, Zuccotti S, Bolognesi M, Viglino P: Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus. Proteins 2006, 62(1):64–79. 10.1002/prot.20703View ArticleGoogle Scholar
- Blaber M, Lindstrom JD, Gassner N, Xu J, Heinz DW, Matthews BW: Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala-->Ser and Val-->Thr substitutions in T4 lysozyme. Biochemistry 1993, 32(42):11363–11373. 10.1021/bi00093a013View ArticleGoogle Scholar
- Zhu BY, Zhou NE, Kay CM, Hodges RS: Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers. Protein Sci 1993, 2(3):383–394.View ArticleGoogle Scholar
- Gromiha MM, Oobatake M, Sarai A: Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins. Biophys Chem 1999, 82(1):51–67. 10.1016/S0301-4622(99)00103-9View ArticleGoogle Scholar
- Querol E, Perez-Pons JA, Mozo-Villarias A: Analysis of protein conformational characteristics related to thermostability. Protein Eng 1996, 9(3):265–271. 10.1093/protein/9.3.265View ArticleGoogle Scholar
- Cox JC, Lape J, Sayed MA, Hellinga HW: Protein fabrication automation. Protein Sci 2007, 16(3):379–390. 10.1110/ps.062591607View ArticleGoogle Scholar
- Zeng G: Sticky-end PCR: new method for subcloning. Biotechniques 1998, 25(2):206–208.Google Scholar
- Gill SC, von Hippel PH: Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 1989, 182(2):319–326. 10.1016/0003-2697(89)90602-7View ArticleGoogle Scholar
- Kabsch W: Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst 1993, 26: 795–800. 10.1107/S0021889893005588View ArticleGoogle Scholar
- Collaborative Computational Project N: The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 1994, 50(Pt 5):760–763. 10.1107/S0907444994003112View ArticleGoogle Scholar
- Jones TA, Zou JY, Cowan SW, Kjeldgaard: Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 1991, 47 ( Pt 2): 110–119. 10.1107/S0108767390010224View 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. 10.1107/S0907444904019158View ArticleGoogle Scholar
- Murshudov GN, Vagin AA, Dodson EJ: Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 1997, 53(Pt 3):240–255. 10.1107/S0907444996012255View ArticleGoogle Scholar
- Laskowski RA MacArthur, M.W., Moss, D.S., Thornton, J.M.: PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 1993, 26: 283–291. 10.1107/S0021889892009944View ArticleGoogle Scholar
- Davis IW, Murray LW, Richardson JS, Richardson DC: MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res 2004, 32(Web Server issue):W615–9. 10.1093/nar/gkh398View ArticleGoogle Scholar
- Kleywegt GJ, Jones TA: Detecting folding motifs and similarities in protein structures. Methods Enzymol 1997, 277: 525–545.View 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.