Abstract
Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- IPTG:
-
Isopropyl thio-β-d-galactoside
- E. coli :
-
Escherichia coli
- SDS–PAGE:
-
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
- EMSA:
-
Electrophoretic mobility shift assay
- MSA:
-
Multiple sequence alignment
- MST:
-
Microscale thermophoresis
- Tris:
-
Tris(hydroxymethyl)aminomethane
- T m :
-
Protein melting temperature
- DBD:
-
DNA binding domain
- HTH:
-
Helix–turn–helix
- DS:
-
Dimerization signal
References
Aerts D, Verhaeghe T, Joosten HJ, Vriend G, Soetaert W, Desmet T (2013) Consensus engineering of sucrose phosphorylase, the outcome reflects the sequence input. Biotechnol Bioeng 110:2563–2572. https://doi.org/10.1002/bit.24940
Alexander P, Fahnestock S, Lee T, Orban J, Bryan P (1992) Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry 31:3597–3603
Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA (2012) Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 78:3458–3464. https://doi.org/10.1128/AEM.07985-11
Azam TA, Ishihama A (1999) Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 274:33105–33113
Becktel WJ, Schellman JA (1987) Protein stability curves. Biopolymers 26:1859–1877
Benitez-Cardoza CG, Rojo-Dominguez A, Hernadez-Arana A (2001) Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein. Biochemistry 40:9049–9058
Berger M, Farcas A, Geertz M, Zhelyazkova P, Brix K, Travers A, Muskhelishvili G (2010) Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU. EMBO Rep 11:59–64. https://doi.org/10.1038/embor.2009.232
Bhowmick T, Ghosh S, Dixit K, Ganesan V, Ramagopal UA, Dey D, Sarma PS, Ramakumar S, Nagarajia V (2014) Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors. Nat Commun 5:4124. https://doi.org/10.1038/ncomms5124
Biltonen RL, Freire E (1978) Thermodynamic characterization of conformational states of biological macromolecules using differential scanning calorimetry. CRC Crit Rev Biochem 5:85–124
Boelens R, Vis H, Vorgias CE, Wilson KS, Kaptein R (1996) Structure and dynamics of the DNA binding protein HU from Bacillus stearothermophilus by NMR spectroscopy. Biopolymers 40:553–559
Bohm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5:191–195
Bonnefoy E, Takahashi M, Yaniv JR (1994) DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA. J Mol Biol 242:116–129. https://doi.org/10.1006/jmbi.1994.1563
Boyko KM, Gorbacheva M, Rakitina T, Korzhenevskiy D, Vanyushkina A, Kamashev D, Lipkin A, Popov V (2015) Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the histone-like HU protein from Spiroplasma melliferum KC3. Acta Crystallogr F Struct Biol Commun 71:24–27. https://doi.org/10.1107/S2053230X14025333
Boyko KM, Rakitina TV, Korzhenevskiy DA, Vlaskina AV, Agapova YK, Kamashev DE, Kleymenov SY, Popov VO (2016) Structural basis of the high thermal stability of the histone-like HU protein from the mollicute Spiroplasma melliferum KC3. Sci Rep 6:36366. https://doi.org/10.1038/srep36366
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Broyles SS, Pettijohn DE (1986) Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch. J Mol Biol 87:47–60
Castaing B, Zelwer C, Laval J, Boiteux S (1995) HU protein of Escherichia coli binds specifically to DNA that contains single-strand breaks or gaps. J Biol Chem 270:10291–10296
Chen C, Ghosh S, Grove A (2004) Substrate specificity of helicobacter pylori histone-like HU protein is determined by insufficient stabilization of DNA flexure points. Biochem J 383:343–351. https://doi.org/10.1042/BJ20040938
Christodoulou E, Vorgias CE (1998) Cloning, overproduction, purification and crystallization of the DNA binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima. Acta Crystallogr D Biol Crystallogr 54:1043–1045
Christodoulou E, Vorgias CE (2002) The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria. Extremophiles 6:21–31
Christodoulou E, Rypniewski WR, Vorgias CE (2003) High-resolution X-ray structure of the DNA-binding protein HU from the hyperthermophilic Thermotoga maritima and the determinants of its thermostability. Extremophiles 7:111–122. https://doi.org/10.1007/s00792-002-0302-7
Cole MF, Gaucher EA (2011) Utilizing natural diversity to evolve protein function: applications towards thermostability. Curr Opin Chem Biol 15:399–406. https://doi.org/10.1016/j.cbpa.2011.03.005
Coste F, Hervouet N, Oberto J, Zelwer C, Castaing B (1999) Crystallization and preliminary X-ray diffraction analysis of the homodimeric form alpha2 of the HU protein from Escherichia coli. Acta Crystallogr D Biol Crystallogr 55:1952–1954
Dame RT, Goosen N (2002) HU, promoting or counteracting DNA compaction? FEBS Lett 529:151–156
Dri AM, Moreau PL, Rouvière-Yaniv J (1992) Role of the histone-like proteins OsmZ and HU in homologous recombination. Gene 120:11–16
Drlica K, Rouviere-Yaniv J (1987) Histonelike proteins of bacteria. Microbiol Rev 51:301–319
Dror A, Shemesh E, Dayan N, Fishman A (2014) Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol. Appl Environ Microbiol 80:1515–1527. https://doi.org/10.1128/AEM.03371-13
Esser D, Rudolph R, Jaenicke R, Bohm G (1999) The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein. J Mol Biol 291:1135–1146. https://doi.org/10.1006/jmbi.1999.3022
Freire E (1995) Differential scanning calorimetry. Methods Mol Biol 40:191–218. https://doi.org/10.1385/0-89603-301-5:191
Garcia-Boronat M, Diez-Rivero CM, Reinherz EL, Reche PA (2008) PVS: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery. Nucleic Acids Res 36:W35–41. https://doi.org/10.1093/nar/gkn211
Garnier N, Loth K, Coste F, Augustyniak R, Nadan V, Damblon C, Castaing B (2011) An alternative flexible conformation of the E. coli HUβ2 protein: structural, dynamics, and functional aspects. Eur Biophys J 40:117–129. https://doi.org/10.1007/s00249-010-0630-y
Ghosh S, Grove A (2004) Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions. J Mol Biol 337:561–571. https://doi.org/10.1016/j.jmb.2004.02.010
Greenfield NJ (1996) Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal Biochem 235:1–10. https://doi.org/10.1006/abio.1996.0084
Grove A (2011) Functional evolution of bacterial histone-like HU proteins. Curr Issues Mol Biol 13:1–12
Grove A, Saavedra TC (2002) The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU. Biochemistry 41:7597–7603
Grove A, Galeone A, Mayol L, Geiduschek EP (1996) Localized DNA flexibility contributes to target site selection by DNA-bending proteins. J Mol Biol 260:120–125
Guo F, Adhya S (2007) Spiral structure of Escherichia coli HUalphabeta provides foundation for DNA supercoiling. Proc Natl Acad Sci USA 13:4309–4314. https://doi.org/10.1073/pnas.0611686104
Hekkelman ML, Te Beek TA, Pettifer SR, Thorne D, Attwood TK, Vriend G (2010) WIWS: a protein structure bioinformatics. Nucleic Acids Res (Web Server Issue). https://doi.org/10.1093/nar/gkq453
Hilser VJ, Townsend BD, Freire E (1997) Structure-based statistical thermodynamic analysis of T4 lysozyme mutants: structural mapping of cooperative interactions. Biophys Chem 64:69–79
Ito Y, Ikeuchi A, Imamura C (2013) Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library. Protein Eng Des Sel 26:73–79. https://doi.org/10.1093/protein/gzs072
Jochens H, Aerts D, Bornscheuer UT (2010) Thermostabilization of an esterase by alignment-guided focussed directed evolution. Protein Eng Des Sel 23:903–909. https://doi.org/10.1093/protein/gzq071
Kabat EA, Wu TT, Bilofsky H (1977) Unusual distributions of amino acids in complementarity-determining (hypervariable) segments of heavy and light chains of immunoglobulins and their possible roles in specificity of antibody-combining sites. J Biol Chem 252:6609–6616
Kamashev D, Rouviere-Yaniv J (2000) The histone-like protein HU binds specifically to DNA recombination and repair intermediates. EMBO J 19:6527–6535. https://doi.org/10.1093/emboj/19.23.6527
Kamau E, Tsihlis ND, Simmons LA, Grove A (2005) Surface salt bridges modulate the DNA site size of bacterial histone-like HU proteins. Biochem J 390:49–55. https://doi.org/10.1042/BJ20050274
Kawamura S, Kakuta Y, Tanaka I, Hikichi K, Kuhara S, Yamasaki N, Kimura M (1996) Glycine-15 in the bend between two alpha-helices can explain the thermostability of DNA binding protein HU from Bacillus stearothermophilus. Biochemistry 35:1195–1200. https://doi.org/10.1021/bi951581l
Kawamura S, Abe Y, Ueda T, Masumoto K, Imoto T, Yamasaki N, Kimura M (1998) Investigation of the structural basis for thermostability of DNA-binding protein HU from Bacillus stearothermophilus. J Biol Chem 273:19982–19987. https://doi.org/10.1074/jbc.273.32.19982
Kim DH, Im H, Jee JG, Jang SB, Yoon HJ, Kwon AR, Kang SM, Lee BJ (2014) β-Arm flexibility of HU from Staphylococcus aureus dictates the DNA-binding and recognition mechanism. Acta Crystallogr D Biol Crystallogr 70:3273–3289. https://doi.org/10.1107/S1399004714023931
Kobryn K, Lavoie BD, Chaconas G (1999) Supercoiling-dependent site-specific binding of HU to naked Mu DNA. J Mol Biol 289:777–784. https://doi.org/10.1006/jmbi.1999.2805
Koh J, Saecker RM, Record MT Jr (2008) DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA–protein complex on intact, linear duplex DNA. J Mol Biol 383:324–346. https://doi.org/10.1016/j.jmb.2008.07.024
Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797. https://doi.org/10.1016/j.jmb.2007.05.022
Kumar S, Tsai CJ, Nussinov R (2003) Temperature range of thermodynamic stability for the native state of reversible two-state proteins. Biochemistry 42:4864–4873. https://doi.org/10.1021/bi027184+
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Larson SM, DiNardo AA, Davidson AR (2000) Analysis of covariation in an SH3 domain sequence alignment: applications in tertiary contact prediction and the design of compensating hydrophobic core substitutions. J Mol Biol 303:433–446. https://doi.org/10.1006/jmbi.2000.4146
Lavoie BD, Shaw GS, Millner A, Chaconas G (1996) Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate. Cell 85:761–771
Lehmann M, Wyss M (2001) Engineering proteins for thermostability, the use of sequence alignments versus rational design and directed evolution. Curr Opin Biotechnol 12:371–375
Lehmann M, Kostrewa D, Wyss M, Brugger R, D'Arcy A, Pasamontes L, van Loon AP (2000a) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng 13:49–57
Lehmann M, Pasamontes L, Lassen SF, Wyss M (2000b) The consensus concept for thermostability engineering of proteins. Biochim Biophys Acta 1543:408–415
Lehmann M, Loch C, Middendorf A, Studer D, Lassen SF, Pasamontes L, van Loon AP, Wyss M (2002) The consensus concept for thermostability engineering of proteins, further proof of concept. Protein Eng 15:403–411
Li S, Waters R (1998) Escherichia coli strains lacking protein HU are UV sensitive due to a role for HU in homologous recombination. J Bacteriol 180:3750–3756
Magliery TJ, Regan L (2004) Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif. J Mol Biol 343:731–745. https://doi.org/10.1016/j.jmb.2004.08.026
Malik M, Bensaid A, Rouviere-Yaniv J, Drlica K (1996) Histone-like protein HU and bacterial DNA topology, suppression of an HU deficiency by gyrase mutations. J Mol Biol 256:66–76. https://doi.org/10.1006/jmbi.1996.0068
McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, Cowley AP, Lopez R (2013) Analysis tool web services from the EMBL-EBI. Nucleic Acids Res 41:W597–W600. https://doi.org/10.1093/nar/gkt376
Miller KM, Jackson SP (2012) Histone marks: repairing DNA breaks within the context of chromatin. Biochem Soc Trans 40:370–376. https://doi.org/10.1042/BST20110747
Miyabe I, Zhang QM, Kano Y, Yonei S (2000) Histone-like protein HU is required for recA gene-dependent DNA repair and SOS induction pathways in UV-irradiated Escherichia coli. Int J Radiat Biol 76:43–49
Mosavi LK, Minor DL Jr, Peng ZY (2001) Consensus-derived structural determinants of the ankyrin repeat motif. Proc Natl Acad Sci USA 99:16029–16034. https://doi.org/10.1073/pnas.252537899
Mouw KW, Rice PA (2007) Shaping the Borrelia burgdorferi genome: crystal structure and binding properties of the DNA-bending protein Hbb. Mol Microbiol 63:1319–1330. https://doi.org/10.1111/j.1365-2958.2007.05586.x
Murphy KP, Freire E (1992) Thermodynamics of structural stability and cooperative folding behavior in proteins. Adv Protein Chem 43:313–361
Nikolova PV, Henckel J, Lane DP, Fersht AR (1998) Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. Proc Natl Acad Sci USA 95:14675–14680
Oberto J, Nabti S, Jooste V, Mignot H, Rouviere-Yaniv J (2009) The HU regulon is composed of genes responding to anaerobiosis, acid stress, high osmolarity and SOS induction. PLoS ONE 4:e4367. https://doi.org/10.1371/journal.pone.0004367
Orfaniotou F, Tzamalis P, Thanassoulas A, Stefanidi E, Zees A, Boutou E, Vlassi M, Nounesis G, Vorgias CE (2009) The stability of the archaeal HU histone-like DNA-binding protein from Thermoplasma volcanium. Extremophiles 13:1–10. https://doi.org/10.1007/s00792-008-0190-6
Padas PM, Wilson KS, Vorgias CE (1992) The DNA-binding protein HU from mesophilic and thermophilic bacilli, gene cloning, overproduction and purification. Gene 117:39–44
Pontiggia A, Negri A, Beltrame M, Bianchi ME (1993) Protein HU binds specifically to kinked DNA. Mol Microbiol 7:343–350
Preobrajenskaya O, Boullard A, Boubrik F, Schnarr M, Rouvière-Yaniv J (1994) The protein HU can displace the LexA repressor from its DNA-binding sites. Mol Microbiol 13:459–467
Privalov PL, Potekhin SA (1986) Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods Enzymol 131:4–51
Ramstein J, Hervouet N, Coste F, Zelwer C, Oberto J, Castaing B (2003) Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure. J Mol Biol 331:101–121
Roth A, Urmoneit B, Messer W (1994) Functions of histone-like proteins in the initiation of DNA replication at oriC of Escherichia coli. Biochimie 76:917–923
Rouviere-Yaniv J, Yaniv M, Germond JE (1979) E. coli DNA binding protein HU forms nucleosome like structure with circular double-stranded DNA. Cell 17:265–274
Ruiz-Sanz J, Filimonov VV, Christodoulou E, Vorgias CE, Mateo PL (2004) Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant. Eur J Biochem 271:1497–1507. https://doi.org/10.1111/j.1432-1033.2004.04057.x
Sagi D, Friedman N, Vorgias CE, Oppenheim AB, Stavans J (2004) Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes. J Mol Biol 341:419–428. https://doi.org/10.1016/j.jmb.2004.06.023
Scopes RK (1974) Measurement of protein by spectrophotometry at 205 nm. Anal Biochem 59:277–282
Shannon CE (1948) The mathematical theory of communication. Bell Syst Tech J 27:379–423. https://doi.org/10.1002/j.1538-7305.1948.tb01338.x
Silva IR, Larsen DM, Jers C, Derkx P, Meyer AS, Mikkelsen JD (2013) Enhancing RGI lyase thermostability by targeted single point mutations. Appl Microbiol Biotechnol 97:9727–9735. https://doi.org/10.1007/s00253-013-5184-3
Steif C, Weber P, Hinz HJ, Flossdorf J, Cesareni G, Kokkinidis M (1993) Subunit interactions provide a significant contribution to the stability of the dimeric four-alpha-helical-bundle protein ROP. Biochemistry 32:3867–3876
Swinger KK, Lemberg KM, Zhang Y, Rice PA (2003) Flexible DNA bending in HU-DNA cocrystal structures. EMBO J 22:3749–3760. https://doi.org/10.1093/emboj/cdg351
Tanaka I, Appelt K, Dijk J, White SW, Wilson KS (1984) 3-Å resolution structure of a protein with histone-like properties in prokaryotes. Nature 310:376–381
Tina KG, Bhadra R, Srinivasan N (2007) PIC: protein interactions calculator. Nucleic Acids Res 35:W473–W476. https://doi.org/10.1093/nar/gkm423
Tominaga T, Nakagawa A, Tanaka I, Kawamura S, Kimura M (1999) High-resolution crystals of the HU mutant K38N from Bacillus stearothermophilus. J Struct Biol 125:86–89. https://doi.org/10.1006/jsbi.1998.4076
Viader-Salvadó JM, Gallegos-López JA, Carreón-Treviño JG, Castillo-Galván M, Rojo-Domínguez A, Guerrero-Olazarán M (2010) Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Appl Environ Microbiol 76:6423–6430. https://doi.org/10.1128/AEM.00253-10
Vis H, Mariani M, Vorgias CE, Wilson KS, Kaptein R, Boelens R (1995) Solution structure of the HU protein from Bacillus stearothermophilus. J Mol Biol 254:692–703. https://doi.org/10.1006/jmbi.1995.0648
Vis H, Vorgias CE, Wilson KS, Kaptein R, Boelens R (1998) Mobility of NH bonds in DNA-binding protein HU of Bacillus stearothermophilus from reduced spectral density mapping analysis at multiple NMR fields. J Biomol NMR 11:265–277
Vriend G (1990) WHAT IF, a molecular modeling and drug design program. J Mol Graph 8:52–56
Wang Q, Buckle AM, Foster NW, Johnson CM, Fersht AR (1999) Design of highly stable functional GroEL minichaperones. Protein Sci 8:2186–2193. https://doi.org/10.1110/ps.8.10.2186
Welfle H, Misselwitz R, Welfle K, Groch N, Heinemann U (1992) Salt-dependent and protein-concentration-dependent changes in the solution structure of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis. Eur J Biochem 204:1049–1055
White SW, Appelt K, Wilson KS, Tanaka I (1989) A protein structural motif that bends DNA. Proteins 5:281–288. https://doi.org/10.1002/prot.340050405
White SW, Wilson KS, Appelt K, Tanaka I (1999) The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus. Acta Crystallogr D Biol Crystallogr 55:801–809. https://doi.org/10.1107/s0907444999000578
Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100. https://doi.org/10.1038/ncomms1093
Wilson KS, Vorgias CE, Tanaka I, White SW, Kimura M (1990) The thermostability of DNA-binding protein HU from Bacilli. Protein Eng Des Sel. https://doi.org/10.1093/protein/4.1.11
Wu TH, Chen CC, Cheng YS, Ko TP, Lin CY, Lai HL, Huang TY, Liu JR, Guo RT (2014) Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design. J Biotechnol 175:1–6. https://doi.org/10.1016/j.jbiotec.2014.01.034
Yu J, Zhou Y, Tanaka I, Yao M (2010) Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics 26:46–52. https://doi.org/10.1093/bioinformatics/btp599
Zhao C, Fu J, Oztekin A, Cheng X (2014) Measuring the Soret coefficient of nanoparticles in a dilute suspension. J Nanopart Res 16(10):2625–2642. https://doi.org/10.1007/s11051-014-2625-6
Zhou HX (2002) Toward the physical basis of thermophilic proteins: linking of enriched polar interactions and reduced heat capacity of unfolding. Biophys J 83:3126–3133. https://doi.org/10.1016/S0006-3495(02)75316-2
Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Längst G, Baaske P (2012) Microscale thermophoresis as a sensitive method to quantify protein, nucleic acid interactions in solution. Methods Mol Biol 815:241–252. https://doi.org/10.1007/978-1-61779-424-7_18
Acknowledgements
We acknowledge technical support by the SPC facility at EMBL Hamburg in the frame of Biostruct-X (EE FP7). We would also like to acknowledge technical assistance from A. Tsoka and N Papandreou for biocomputing in our department. F. S. has been supported in part by the ARISTEIA I program (Grant Number 1125), administered by the General Secretariat of Research and Technology of Greece, co-financed by the European Social Fund and the State of Greece. The financial support of "The National Research Infrastructures on Integrated Structural Biology, Drug Screening Efforts and Drug target functional characterization” with the acronym “INSPIRED" and code (MIS) 5002550 from the Greek Government is also acknowledged.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by L. Huang.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
792_2020_1154_MOESM1_ESM.pdf
Figure 1 Supplement The sequences were aligned using the ClustalO software, available on-line, and the consensus sequence of HUs was determined from the most commonly occurring amino acid in each position of the MSA (PDF 71 kb)
792_2020_1154_MOESM2_ESM.pdf
Figure 2 Supplement The amino acid primary structure of the consensus HUBest protein and the codon optimized gene sequence for E. coli of the corresponding hubest gene (PDF 41 kb)
792_2020_1154_MOESM3_ESM.docx
Figure 3 Supplement Sequence alignment of the HUBest with the mesostable HUBsu, HUStaam thermostable HUBst and the hyperthermostable HUTmar. The amino acids that are related with the thermostability are indicated by *. Amino acids, which are discussed for the binding to DNA, are indicated by + (DOCX 15 kb)
792_2020_1154_MOESM4_ESM.docx
Table 1 Supplement The Uniprot entries of the 40 HU that comprise the data set for the determination of the consensus sequence of HUBest. The identity score between HUBest and each individual HU entry is also included (DOCX 78 kb)
792_2020_1154_MOESM5_ESM.docx
Table 2 Supplement Comparison of the molecular interactions and geometry between selected HUs. A comparison of the molecular interactions between the HUBest (model), HU from Staphylococcus aureus HUStaam (4QJU), HU from Bacillus stearothermophilus HUBst (1HUU) and HU from Thermotoga maritima HUTmar (1B8Z) calculated according the default parameters of the server: http://pic.mbu.iisc.ernet.in/. The computations of the surfaces were carried out with PISA server: http://www.ebi.ac.uk/pdbe/pisa/. The computations of the cavities were carried out with POCASA (http://altair.sci.hokudai.ac.jp/g6/service/pocasa/) (DOCX 71 kb)
Rights and permissions
About this article
Cite this article
Georgoulis, A., Louka, M., Mylonas, S. et al. Consensus protein engineering on the thermostable histone-like bacterial protein HUs significantly improves stability and DNA binding affinity. Extremophiles 24, 293–306 (2020). https://doi.org/10.1007/s00792-020-01154-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-020-01154-4