Mutation of exposed hydrophobic amino acids to arginine to increase protein stability
© Strub et al; licensee BioMed Central Ltd. 2004
Received: 25 February 2004
Accepted: 13 July 2004
Published: 13 July 2004
One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.
In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion.
Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.
Acetylcholinesterase (AChE, EC 188.8.131.52) is a serine hydrolase, which catalyzes the hydrolysis of the neurotransmitter acetylcholine. This enzyme is irreversibly inhibited by organophosphate and carbamate pesticides leading to its use in biosensors to detect traces of these compounds in environment. Drosophila AChE was found to be the most sensitive enzyme when compared to enzymes of non-insect origin and in-vitro-mutagenesis was used to select enzymes up to 300-fold more sensitive [1, 2]. But like most enzymes from mesophilic organisms, Drosophila AChE is not stable, and this instability precludes its utilization in biosensors. It can be stabilized by adding some molecules in the solution such as reversible inhibitors, polyethylene glycol or protein, provoking protein-protein interactions. Alternatively, stabilization may also be achieved by encapsulation in liposomes [3, 7]. Another way to stabilize the enzyme is to use in vitro mutagenesis to modify the primary structure of the protein . This method could have the additional advantage of stabilizing the enzyme during its synthesis leading to higher production and higher purification yields.
Irreversible denaturation of AChE at room temperature can be minimized by increasing the protein concentration in the sample, either by increasing the enzyme concentration or by addition of another protein such as BSA . This suggested that denaturation occurred by interaction of the hydrophobic region at the surface of AChE with tube walls or air-solvent interface. Addition of protein in the solution would compete with hydrophobic surfaces and protect the enzyme. We thus hypothesized that decreasing the hydrophobicity at the surface of the protein may have some stabilizing effects.
Several examples showed that the change of hydrophobicity to hydrophilicity of amino-acid residues exposed to the solvent at the surface of proteins is an efficient stategy to stabilize proteins: i) Analysis of protein sequences showed a strong bias for hydrophilic residues and against large hydrophobic residues at most surface positions , ii) Mutagenesis showed that hydrophilic amino acids at surface positions is stabilizing Mutagenesis showed that hydrophilic amino acids at surface positions have stabilizing effect, while placing a hydrophobic residue in a solvent-exposed position causes destabilization [10–12], iii) The proportion of hydrophobic residues at the surface of proteins from hyperthermophilic species was found to be reduced compared to the proportion in their mesophilic counterparts [13–16], however, this observation is under debate .
Here we tested this strategy by mutating several hydrophobic residues scattered at the surface of Drosophila AChE to arginine. Hydrophobic residues were chosen by visual examination of the structure and arginine was chosen because the guanidinium group is the most polar of all the common amino-acid residues found in proteins.
Results and discussion
Effect of mutations on protein production
Effect of mutations on protein stability
half-life (t 50 ) of the wild type protein.
8 +/- 1.7
4 M Urea
11 +/- 1.6
1.45 +/- 0.18
0.1 mg/ml pronase
14.4 +/- 2.2
The effect of mutations on stability was homogeneous, a mutation either destabilizes or stabilizes the protein since we never found a mutation which significantly stabilizes the protein for one agent and significantly destabilizes it for another.
Some mutations destabilized the protein. The presence of hydrophobic residues at the surface may have stabilization properties by providing a shield from penetrating water molecules . Or mutation to Arg may disrupt a hydrophobic bond. V56 and L57 are in hydrophobic contact with Val 14, mutation to Arg may disrupt this bond. Likewise, Ile 5 faces Gly 16 and this interaction may maintain the loop conformation.
Protein production and purification
cDNA encoding wild type drosophila AChE and mutant were expressed with the baculovirus system . This enzyme is a dimer anchor to the membrane via a glycolipid . We expressed a soluble dimeric form obtained by deletion of the hydrophobic peptide, precursor of the glycolipid anchor at the C-terminal end . Furthermore, the enzyme was deleted of the loop from amino-acids 103 to 136 which has been replaced by 3 histidines. Secreted AChE was purified to homogeneity using the following steps: ammonium sulfate precipitation, ultrafiltration with a 10 kDa cutoff membrane, NTA-nickel chromatography, affinity chromatography with procainamide as ligand and gel filtration . Activity was recorded at 25°C in 25 mM phosphate buffer pH 7, with 1 mM acetylthiocholine iodide as substrate using the method of Ellman et al. . Mutations did not alter the specific activity of the proteins. Molecular modeling of mutated residues was performed from the structure solved by Harel et al. [, ref 1QO9] by homology using Swiss model server available at http://expasy.org/swissmod/. Residue numbering followed that of the mature protein.
One liter containing 106 Sf9 cells was infected with more than 107 virus. After four days incubation at 28°C, the cells were lyzed by adding Triton X-100 (0.1%). Amount of AChE was estimated by active site titration using an irreversible inhibitor, chlorpyriphos oxon . The mean of at least five independent productions was compared to the production of the wild type enzyme.
All denaturation experiments were performed with 10 picomoles of enzyme in one ml 25 mM phosphate buffer pH7 at 25°C. AChE was incubated in denaturing conditions. Aliquots were taken at regular intervals, diluted 20 x to stop the action of the denaturing agent and the remaining activity was measured.
To analyze thermosensitivity, enzymes were incubated at 50°C with 1 mg/ml Bovine Serum Albumin in the buffer. Before recording the remaining activity, aliquots were mixed with cold buffer chilled on ice and the solution was incubated at 25°C for ten minutes to eliminate the reversible component . For urea denaturation, unfolding of AChE was induced by adding 4 M urea into the incubation buffer. The effect of organic solvent was followed by incubation of the enzyme in 20% acetonitrile. The effect of protease sensitivity was determined by incubation of AChE with 0.1 mg/ml pronase.
Three to nine batches of each mutant were produced and purified. Three repeats were performed for each batch and each denaturing agent. Significance of difference observed in stability was tested using the Mann Whitney test.
Coulombic interaction estimation
In order to estimate the effects of charge distribution on each of the mutants, the contributions from electrostatic interactions were extracted from the total energy of the minimized structures of the molecules. The minimization was performed with the GROMACS software , using the simulated annealing protocol. Position restraints were imposed on the whole molecule with the exception of the mutated residue, which was allowed to adopt the energetically favorable configuration. The Coulomb's interactions were normalized by subtracting the value obtained for the wild type protein and by rescaling the numbers by 80, to take into account the dielectric constant of water.
List of abbreviations
This research was supported by grants from the European Community (ACHEB, QLK3-CT-2000-00650 and SAFEGUARD, QLK3-CT-2000-000481) and from DGA (PEA 99CO029).
- Villatte F, Marcel V, Estrada-Mondaca S, Fournier D: Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens Bioelectron. 1998, 13: 157-162. 10.1016/S0956-5663(97)00108-5.View ArticlePubMedGoogle Scholar
- Boublik Y, Saint-Aguet P, Lougarre A, Arnaud M, Villatte F, Estrada-Mondaca S, Fournier D: Acetylcholinesterase engineering for detection of insecticide residues. Protein Engin. 2002, 15: 43-50. 10.1093/protein/15.1.43.View ArticleGoogle Scholar
- Payne CS, Saeed M, Wolfe AD: Ligand stabilization of cholinesterases. Biochim Biophys Acta. 1989, 999: 46-51. 10.1016/0167-4838(89)90028-9.View ArticlePubMedGoogle Scholar
- Burgess SK, Oxendine SL: Thermal inactivation of butyrylcholinesterase and acetylcholinesterase. J Prot Chem. 1983, 12: 651-658.View ArticleGoogle Scholar
- Wilson EJ, Massoulié J, Bon S, Rosenberry TL: The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant. FEBS Lett. 1996, 379: 161-164. 10.1016/0014-5793(95)01504-3.View ArticlePubMedGoogle Scholar
- Estrada-Mondaca S, Fournier D: Stabilization of recombinant Drosophila acetylcholinesterase. Prot Exp Purif. 1998, 12: 166-172. 10.1006/prep.1997.0831.View ArticleGoogle Scholar
- Nasseau M, Boublik Y, Meier W, Winterhalter M, Fournier D: Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation and protects against proteolytic degradation. Biotech Bioeng. 2001, 75: 615-618. 10.1002/bit.10074.View ArticleGoogle Scholar
- Fremaux I, Mazères S, Brisson-Lougarre A, Arnaud M, Ladurantie C, Fournier D: Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine. BMC Biochemistry. 2002, 3: 21-10.1186/1472-2091-3-21.PubMed CentralView ArticlePubMedGoogle Scholar
- Bashford D, Chothia C, Lesk AM: Determinants of a protein fold. Unique features of the globin amino acid sequences. J Mol Biol. 1987, 196: 199-216.View ArticlePubMedGoogle Scholar
- Reidhaar-Olson JF, Sauer RT: Functionally acceptable substitutions in two alpha-helical regions of lambda repressor. Proteins. 1990, 7: 306-316.View ArticlePubMedGoogle Scholar
- Schwehm JM, Kristyanne ES, Biggers CC, Stites WE: Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease. Biochemistry. 1998, 37: 6939-6948. 10.1021/bi9725069.View ArticlePubMedGoogle Scholar
- Wigley DB, Clarke AR, Dunn CR, Barstow DA, Atkinson T, Chia WN, Muirhead H, Holbrook JJ: The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of a water-accessible hydrophobic surface. Biochim Biophys Acta. 1987, 916: 145-148. 10.1016/0167-4838(87)90221-4.View ArticlePubMedGoogle Scholar
- Auerbach G, Huber R, Grättinger M, Zaiss K, Schurig H, Jaenicke R, Jacob U: Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: the crystal structure at 2.1 Å resolution reveals strategies for intrinsic protein stabilization. Structure. 1997, 5: 1475-1483. 10.1016/S0969-2126(97)00297-9.View ArticlePubMedGoogle Scholar
- Argos P, Rossman RG, Grau , Zuber H, Franck G, Tratschin JD: Thermal stability and protein structure. Biochemistry. 1979, 18: 5698-5703.View ArticlePubMedGoogle Scholar
- Vogt G, Argos P: Protein thermal stability: hydrogen bonds or internal packing?. Fold Des. 1997, 2: S40-S46.View ArticlePubMedGoogle Scholar
- Vogt G, Woell S, Argos P: Protein thermal stability, hydrogen bonds, and ion pairs. J Mol Biol. 1997, 269: 631-643. 10.1006/jmbi.1997.1042.View ArticlePubMedGoogle Scholar
- Kumar S, Tsai CJ, Nussinov R: Factors enhancing protein thermostability. Protein Eng. 2000, 13: 179-91. 10.1093/protein/13.3.179.View ArticlePubMedGoogle Scholar
- Pakula AA, Sauer RT: Reverse hydrophobic effects relieved by amino-acid substitution at a protein surface. Nature. 1990, 344: 363-364. 10.1038/344363a0.View ArticlePubMedGoogle Scholar
- Rennell D, Bouvier SE, Hardy LW, Poteete AR: Systematic mutation of bacteriophage T4 lysozyme. J Mol Biol. 1991, 222: 67-88.View ArticlePubMedGoogle Scholar
- Perl D, Mueller U, Heinemann U, Schmid FX: Two exposed amino acid residues confer thermostability on a cold shock protein. Nat Struct Biol. 2000, 7: 380-383. 10.1038/75151.View ArticlePubMedGoogle Scholar
- Pedone E, Saviano M, Rossi M, Bartolucci S: A single point mutation (Glu85Arg) increases the stability of the thioredoxin from Escherichia coli. Protein Eng. 2001, 14: 255-260. 10.1093/protein/14.4.255.View ArticlePubMedGoogle Scholar
- Van den Burg B, Dijkstra BW, Vriend G, Van Der Vinne B, Venema G, Eijsink VGH: Protein stabilization by hydrophobic interactions at the surface. Eur J Biochem. 1994, 220: 981-985.View ArticlePubMedGoogle Scholar
- Perutz MF, Raidt S: Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2. Nature. 1975, 255: 256-259.View ArticlePubMedGoogle Scholar
- Jaenicke R, Bohm G: The stability of proteins in extreme environments. Curr Opin Struct Biol. 1998, 8: 738-748. 10.1016/S0959-440X(98)80094-8.View ArticlePubMedGoogle Scholar
- Pace CN: Single surface stabilizer. Nat Struct Biol. 2000, 7: 345-346. 10.1038/75100.View ArticlePubMedGoogle Scholar
- Sanchez-Ruiz JM, Makhatadze GI: To charge or not to charge?. Trends Biotechnol. 2001, 19: 132-135. 10.1016/S0167-7799(00)01548-1.View ArticlePubMedGoogle Scholar
- Spector S, Wang M, Carp SA, Robblee J, Hendsch ZS, Fairman R, Tidor B, Raleigh DP: Rational modification of protein stability by the mutation of charged surface residues. Biochemistry. 2000, 39: 872-879. 10.1021/bi992091m.View ArticlePubMedGoogle Scholar
- Grimsley GR, Shaw KL, Fee LR, Alston RW, Huyghues-Despointes BM, Thurlkill RL, Scholtz JM, Pace CN: Increasing protein stability by altering long-range coulombic interactions. Protein Sci. 1999, 8: 1843-1849.PubMed CentralView ArticlePubMedGoogle Scholar
- Chaabihi H, Fournier D, Fedon Y, Bossy JP, Devauchelle G, Cerutti M: Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculovirus. Biochem Biophys Res Commun. 1994, 203: 734-742. 10.1006/bbrc.1994.2243.View ArticlePubMedGoogle Scholar
- Fournier D, Berge JB, Cardoso de Almeida ML, Bordier C: Acetylcholinesterases from Musca domestica and Drosophila melanogaster brain are linked to membranes by a glycophospholipid anchor sensitive to an endogenous phospholipase. J Neurochem. 1988, 50: 1158-1163.View ArticlePubMedGoogle Scholar
- Fournier D, Mutero A, Rungger D: Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes. Eur J Biochem. 1992, 203: 513-519.View ArticlePubMedGoogle Scholar
- Ellman GL, Courtney KD, Andres KD, Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmac. 1961, 7: 88-95. 10.1016/0006-2952(61)90145-9.View ArticleGoogle Scholar
- Harel M, Kryger G, Rosenberry TL, Mallender WD, Lewis T, Fletcher RJ, Guss JM, Silman I, Sussman JL: Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors. Protein Sci. 2000, 9: 1063-1072.PubMed CentralView ArticlePubMedGoogle Scholar
- Charpentier A, Menozzi P, Marcel V, Villatte F, Fournier D: A method to estimate acetylcholinesterase active sites and turnover in insects. Anal Biochem. 2000, 285: 76-81. 10.1006/abio.2000.4738.View ArticlePubMedGoogle Scholar
- Pavlic MR, Alif M, Bozic M, Stojan J: On the stability of some enzymes under various experimental conditions. Period Biolog. 1991, 93: 211-215.Google Scholar
- Lindahl E, Hess B, van der Spoel D: GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model. 2001, 7: 306-317.Google Scholar
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