YABIO 11717
No. of Pages 3, Model 5G
15 May 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7
Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase
5 8 9 10
11 1 6 3 2 14 15 16 17 18 19 20 21 22 23 24 25
Katherine M. Hoffmann ⇑, Kaitlin M. Wood, Alysha D. Labrum, Dave K. Lee, Ingmar M. Bolinger, Mary E. Konis, Adam G. Blount, Gregory A. Prussia, Monica M. Schroll, Jeffrey M. Watson Department of Chemistry and Biochemistry, Gonzaga University, Spokane, WA 99258, USA
a r t i c l e
i n f o
Article history: Received 14 January 2014 Received in revised form 14 April 2014 Accepted 17 April 2014 Available online xxxx Keywords: b-Carbonic anhydrase Haemophilus influenzae Immobilized metal affinity purification Surface histidines Endogenous nickel affinity
a b s t r a c t Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques. Ó 2014 Elsevier Inc. All rights reserved.
27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Immobilized metal affinity chromatography has achieved widespread use due to the many advantages of the technique, including ease of imidazole elution and significant purification in one step from protein expression systems [1]. Drawbacks to the method are few but include interference of tags with the quaternary structure and non-native random coil interfering with downstream techniques. To accommodate these issues, additional extensions or protease sites may be reengineered, but these introduce a nondesirable addition of random coil or a new experimental step in purification. The tag may be moved to the other terminus of the protein, but there are no further termini available if that one is also problematic. The presence of a half-dozen identified proteins in Escherichia coli with endogenous nickel affinity [2] suggests that metal affinity may be engineered on the surface of a protein instead of in a terminal tag. Logically, only two histidines might be required given that four of the six ligand positions on Ni2+ ions are commonly occupied by the solid phase ligands [1]. The optimal distance between a dyad pair of histidines can be extrapolated from inorganic structures and might be expected to be between 4 and 7 Å given the rotational range of motion around the CaACb bond in histidine. Tags are commonly engineered with six histidines on a terminus, however, and proteins with endogenous nickel affinity have between 3 and 6 histidines in a cluster, potentially providing a number of vicinal ligand pairs. Delineating the parameters of ⇑ Corresponding author. Fax: +1 509 313 5804. E-mail address: [email protected] (K.M. Hoffmann).
histidine location on the surface of proteins required to bind metal would define a broadly applicable extension of the metal affinity technique, expanding the number of possible metal affinity sites, minimizing the addition of random coil and non-native residues, and eliminating the need to remove affinity tags after purification. In addition, surface metal affinity could be used for reporter labeling or crystallography phasing. Before a general method can be developed, however, the characteristics present in proteins with endogenous metal affinity must be successfully applied to a similar system and the concept must be proven. Carbonic anhydrases (carbonate hydrolyase, EC 4.2.1.1) are zinc metalloenzymes that catalyze the reversible interconversion of CO2 and bicarbonate:
CO2 þ H2 O¡HCO3 þ Hþ :
ð1Þ
E. coli carbonic anhydrase (ECCA)1 is a member of the b-form of carbonic anhydrases, widely distributed in bacteria, yeast, and plant chloroplasts, serendipitously identified when it is bound persistently to a nickel column without any engineered tag [3]. Although all forms of carbonic anhydrases have convergently evolved to use metal ions in their catalytic mechanism, none uses nickel [4]. ECCA nevertheless binds so tightly to an Ni2+ column that 250 to 300 mM imidazole is needed to elute it off of the metal sites, comparable to the amount 1 Abbreviations used: ECCA, Escherichia coli carbonic anhydrase; ICP–OES, inductively coupled plasma–optical emission spectroscopy; HICA, Haemophilus influenzae carbonic anhydrase; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
http://dx.doi.org/10.1016/j.ab.2014.04.020 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
62 63 64 65 66 67 68 69 70 71 72 73 74
75 77 78 79 80 81 82 83 84 85
YABIO 11717
No. of Pages 3, Model 5G
15 May 2014 2 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151
Notes & Tips / K.M. Hoffmann et al. / Anal. Biochem. xxx (2014) xxx–xxx
needed to compete off a histidine-tagged protein [3]. There is no loss of ECCA activity using this purification method, and no nickel ions are retained in the protein itself based on inductively coupled plasma– optical emission spectroscopy (ICP–OES) measurements [3]. ECCA, like other b-carbonic anhydrases, has a dimer of dimers quaternary structure. The tetramerization interface between dimers is partly solvent exposed and contains two symmetry-related clusters of histidines. Each cluster includes six histidines from two chains (His72, His122, and His160 from one chain; His720 , His1220 , and His1600 from the other chain). The histidines are between 5 and 7 Å apart in the same chain, although the interchain distance between His72 and His1220 is closer at between 3.5 and 4.5 Å. Because nickel(II)–histidine coordination distances have been observed in protein structures at 1.8 to 2.4 Å [5], these side chains were hypothesized to be within the sphere of rotation necessary to optimize nickel coordination (see Fig. S1 in online Supplementary material) [2]. Haemophilus influenzae carbonic anhydrase (HICA), although a bcarbonic anhydrase family member sharing 38% identity, quaternary structure, and activity profile similar to ECCA, does not share the endogenous surface metal affinity and only partially conserves the cluster of histidines in the tetramerization interface (His72, His122, and Arg160), even though the residues overlay well. Mutating Arg160 to histidine in HICA sufficiently recreated ECCA’s metal affinity and allowed purification under similar conditions. Optimizing Arg160His HICA’s purification to minimize secondary retention during the binding and wash steps meant that the protein variant eluted earlier in the gradient but similarly achieved greater than 90% purity. Steady-state kinetic activity and oligomerization state were similar to wild-type HICA, establishing that the surface mutations did not influence the overall structure or function of the protein. This proof-of-concept system is the first step toward establishing the parameters for a general engineered surface metal affinity method. Arg160His was constructed using megaprimer PCR [6] with Pfu Ultra (Stratagene) polymerase and commercial oligonucleotides (Integrated DNA Technologies). A mutant oligonucleotide (50 Q1 CAATCTAGGGCACACATCAATTGT-30 ) was paired with the 50 terminal oligonucleotide primer PHI2X (50 -TGCCTGCAGTTATTATGTAT Q2 TTTCAAGATG-30 ) in the first polymerase chain reaction (PCR) to give a 226-bp product. This PCR product was used as a megaprimer in a second PCR with the 30 oligonucleotide primer PHI1QC (50 TGCCCATGGATAAAATTAAACAACTCTTT-30 ) to create the final mutated HICA gene. The final PCR product was used as a megaprimer in a QuikChange protocol. The Arg160His HICA gene was sequenced (Clemson University Sequencing Institute) to verify the introduction of the correct mutations. Expression of variant HICA proteins was as described previously [7]. Purification of ECCA and HICA proteins was as described for the wild-type ECCA protein except that detergent was omitted and 20 mM imidazole was used in the wash step for Arg160His HICA. Crude homogenates of overexpressed protein were bound to a nickel column (HisTrap HP, GE Healthcare) in buffer A (20 mM Tris–HCl [pH 8.0], 0.1 M NaCl, and 10 mM imidazole), washed, and purified to homogeneity using an AKTA FPLC (fast protein liquid chromatography) system (GE Healthcare) and a linear gradient of 20 to 500 mM imidazole. Fractions were pooled and desalted using a HiPrep 26/10 desalting column (GE Healthcare) and 20 mM Tris–HCl (pH 8.0) and 0.1 M NaCl buffer. Native oligomerization state was confirmed using the same buffer and a Sephadex 200 30/600 gel exclusion chromatography column (GE Healthcare). All proteins were quantified by absorbance at 280 nm [8], monomeric size was confirmed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE), and purity was estimated by densitometry analysis using the program ImageJ [9]. To explore whether the kinetic activity of Arg160His HICA was influenced by the mutation, steady-state kinetics experiments at
pH 9.0 were performed for both the HICA variant and wild-type ECCA, as described previously. Saturated solutions of CO2 were prepared by bubbling CO2 gas into water in a vessel maintained at 25.0 ± 0.1 °C, and dilutions were prepared by coupling two gas-tight syringes as described by Khalifah [10]. CO2 concentrations were calculated based on a 33.8-mM saturated solution at 25.0 °C [11]. All steady-state kinetic measurements were made at 25.0 °C using a Hi-Tech SF-61DX2 stopped-flow spectrophotometer and were carried out in the presence of 250 mM Na2SO4 for maximum enzyme stability and activity in dilute solution. Initial rates of CO2 hydration were measured using the changing pH indicator method described
Fig.1. (A) Normalized chromatography of Arg160His HICA and controls. 0 volume is after a 5-column-volume wash with 10 mM (ECCA and HICA) or 20 mM (Arg160His HICA) imidazole concentration and indicates the start of the gradient. ECCA (green dashed line) elutes at 11.2 ml and 278 mM imidazole. Wild-type HICA (red solid line) shows no retention on the nickel column. The increasing baseline is likely due to contaminants in the imidazole salt. Arg160His HICA (blue solid line) elutes at 7.7 ml and 190 mM imidazole. The linear imidazole gradient is shown as a black dotted line with the axis plotted on the right and does not reflect differing concentrations of imidazole in the wash step. (B) Representative gel exclusion chromatography elution with the x axis reflecting the retention volume (below the axis) and the retention volume of the standards used to estimate the molecular mass of the oligomer. Arg160His HICA elutes at a volume consistent with the wildtype biological tetramer. (C) SDS–PAGE gel of Arg160His HICA purification steps. Lane 1: soluble lysate from overexpression; lane 2: a fraction immediately preceding the main elution peak; lane 3: a fraction from the main elution peak with a distinct band at approximately 26 kDa; lane 4: from the shoulder peak at approximately 278 kDa; lane 5: pooled gel exclusion peak with a main monomer band at 26 kDa. (For interpretation of the references to color in this figure legend and the text, the reader is referred to the Web version of this article.)
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
152 153 154 155 156 157 158 159 160 161 162
YABIO 11717
No. of Pages 3, Model 5G
15 May 2014 3
Notes & Tips / K.M. Hoffmann et al. / Anal. Biochem. xxx (2014) xxx–xxx Table 1 Chromatography retention of Arg160His HICA as well as positive (ECCA) and negative (HICA) controls. Protein
Retention volume peak (ml)
[Imidazole] peak (mM)
Purity (%)
kcat (ms1)
kcat/Km (lM1 s1)
ECCA HICA Arg160His HICA
11.2 0 7.7
278 0 190
92.2 0 90.31
77 ± 7 69 ± 29a 37 ± 3
2.4 ± 0.7 4.3 ± 0.8a 2.7 ± 0.2
Note. Retention volume is calculated relative to the imidazole gradient on a 5-ml column, and a retention volume of 0 (zero) indicates that the lysate or protein was not on the column when the gradient was initiated. Purity was estimated from densitometry of SDS–PAGE gels using the program ImageJ [9]. Kinetic values of kcat and kcat/Km for HICA wild-type and variants were determined at pH 9.0 in reaction conditions of 40 mM 1,2-dimethylimidazole, 58 lM m-cresol purple, 1 lM ethylenediaminetetraacetic acid (EDTA), and 250 mM sodium sulfate. Reaction conditions for ECCA at pH 9.0 were 40 mM bicine, 25 lM m-cresol purple, 2.5 mM EDTA, and 250 mM sodium sulfate. a Wild-type HICA kinetic constants were originally reported by Cronk and coworkers [7]. 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
previously [10–12]. Values of kcat (reported on a per subunit basis) and Km were determined by nonlinear least squares fits to v/[E] versus [CO2] data using Origin 7.0 (Microcal). We have successfully engineered surface histidines for the nickel purification of HICA using an Arg160His mutation to complete a cluster of histidine residues (His72, His1220 , and His160) in ECCA that was partially conserved in HICA. However, the core of the cluster, His122 and His72 (and their symmetry mates), did not have metal affinity on their own (Fig. 1A, wild-type HICA control). Previous crystallization of HICA variants has revealed solvent phosphate molecules coordinated by several residues in the tetramerization interface, including Arg160 [13], suggesting that that the 160 position is a key solvent-exposed histidine. The purification of Arg160His HICA based on the previously published ECCA purification buffers eluted the main peak at a similar concentration to ECCA (278 mM at the peak; Fig. 1A, green line) but had significant contamination when washed with a 10-mM imidazole concentration (data not shown). Wild-type HICA protein had no affinity for the nickel column (Fig. 1A, red line). To optimize the purification of Arg160His HICA, we increased the initial imidazole concentration to 20 mM and observed the elution of a specifically bound protein at 190 mM in the gradient (Fig. 1A, blue line). Using the optimized purifications, we purified Arg160His HICA to Q3 greater than 90% purity with a single step (Fig. 1C and Table 1). To confirm that the Arg160His mutation did not alter the native chemistry of HICA, we performed steady-state kinetics and found that kcat = 37 ± 3 ms1 and kcat/Km = 2.7 ± 0.2 lM1 s1 (Table 1). These values are similar to wild-type numbers and well within the range of nonconsequential HICA variant activity observed preQ3 viously. Because the Arg160His mutation was located in the oligomerization interface, we confirmed that Arg160His HICA eluted at a size consistent with a tetramer (122 ± 24 kDa) using gel exclusion chromatography (Fig. 1B). These experiments establish that the location and number of mutations does not disrupt the native structure or behavior of the protein. Using published crystal structure PDB 2A8D, we have modeled the Arg160His mutation (Fig. S1) and examined the ability of the residues to make the necessary ligand distances to a metal as well as the solvent accessibility of the cluster. Rotation of the nearby His72 and His1220 about the CaACb bond easily allowed His72 and Arg160His to rotate to within 3.3 Å of each other, sufficient to simultaneously accommodate approximately 2-Å ligand distances to a nickel ion with reasonable angles and good solvent exposure. The location of Arg160His on the edges of the buried histidine clusters means that there are four possible dyad nickel binding sites per HICA tetramer. Whether the metal affinity is due exclusively to multiple dyad histidine pairs or whether the rest of the cluster plays activating roles is not clear, but either hypothesis would be consistent with most other identified proteins with endogenous nickel affinity presenting clusters of at least three exposed proximal histidines [2]. Compared with the published three-column purification scheme (cation exchange, hydrophobic exchange, and gel exclusion chromatography) for HICA [9], metal affinity purification is a substantial improvement in time and resources. Beyond the advan-
tage for the carbonic anhydrases, however, this method is a proofof-concept that may be extended to numerous other systems in which a terminal histidine tag is undesirable such as for structural studies requiring a native-like tertiary structure. Vicinal histidines or designed surface clusters also have the extended potential to be used for reporter labeling or deliberate metal ion binding to a protein’s surface. We have shown that histidine-based metal affinity may be engineered onto the surface of a protein rather than restricting an engineered tag to one of two termini. To extend this work to a general method, we will repeat the histidine mutations in a more completely surface-exposed area of the protein and establish the minimal histidine character needed for nickel affinity as well as the stoichiometry of metal binding via ICP–OES. Future work will also explore the role of extra histidines beyond the minimum dyad pair to create optimal surface metal affinity.
217
Acknowledgments
232
The authors gratefully acknowledge the helpful contributions of Roger S. Rowlett and also thank Jeffrey Cronk, Hannah Maul, Dana Walters, and the students of Chem 443L for their assistance.
233
Appendix A. Supplementary data
236
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.020.
237
References
239
[1] J. Crowe, B.S. Masone, J. Ribbe, One-step purification of recombinant proteins with the 6His tag and Ni-NTA resin, Mol. Biotechnol. 4 (1995) 247–258. [2] V.M. Bolanos-Garcia, O.R. Davies, Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography, Biochim. Biophys. Acta 1760 (2006) 1304–1313. [3] J.D. Cronk, J.W. O’Neill, M.R. Cronk, J.A. Endrizzi, K.Y.J. Zhang, Cloning, crystallization, and preliminary characterization of a -carbonic anhydrase from Escherichia coli, Acta Crystallogr. D 56 (2000) 1176–1179. [4] R.S. Rowlett, Structure and catalytic mechanism of the-carbonic anhydrases, Biochim. Biophys. Acta 2010 (1804) 362–373. [5] R. Balakrishnan, N. Ramasubbu, K.I. Varughese, R. Parthasarathy, Crystal structures of the copper and nickel complexes of RNase A: metal-induced interprotein interactions and identification of a novel copper binding motif, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 9620–9625. [6] G. Sarkar, S.S. Sommer, The ‘‘megaprimer’’ method of site-directed mutagenesis, BioTechniques 8 (1990) 404–407. [7] J.D. Cronk, R.S. Rowlett, K.Y.J. Zhang, C.K. Tu, J.A. Endrizzi, J. Lee, P.C. Gareiss, J.R. Preiss, Identification of a novel noncatalytic bicarbonate binding site in eubacterial-carbonic anhydrase, Biochemistry 45 (2006) 4351–4361. [8] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem. 182 (1989) 319–326. [9] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9 (2012) 671–675. [10] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase: I. Stop-flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971) 2561–2573. [11] A.F. Ghannam, W. Tsen, R.S. Rowlett, Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2, J. Biol. Chem. 261 (1986) 1164–1169. [12] R.S. Rowlett, N.J. Gargiulo, F.A. Santoli, J.M. Jackson, A.H. Corbett, Activation and inhibition of bovine carbonic anhydrase III by dianions, J. Biol. Chem. 266 (1991) 933–941. [13] R. Rowlett, C. Tu, J. Lee, A. Herman, Allosteric site variants of Haemophilus influenzae b-carbonic anhydrase, Biochemistry 48 (2009) 6146–6156.
240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
218 219 220 221 222 223 224 225 226 227 228 229 230 231
234 235
238
273
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
No. of Pages 3, Model 5G
15 May 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7
Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase
5 8 9 10
11 1 6 3 2 14 15 16 17 18 19 20 21 22 23 24 25
Katherine M. Hoffmann ⇑, Kaitlin M. Wood, Alysha D. Labrum, Dave K. Lee, Ingmar M. Bolinger, Mary E. Konis, Adam G. Blount, Gregory A. Prussia, Monica M. Schroll, Jeffrey M. Watson Department of Chemistry and Biochemistry, Gonzaga University, Spokane, WA 99258, USA
a r t i c l e
i n f o
Article history: Received 14 January 2014 Received in revised form 14 April 2014 Accepted 17 April 2014 Available online xxxx Keywords: b-Carbonic anhydrase Haemophilus influenzae Immobilized metal affinity purification Surface histidines Endogenous nickel affinity
a b s t r a c t Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques. Ó 2014 Elsevier Inc. All rights reserved.
27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Immobilized metal affinity chromatography has achieved widespread use due to the many advantages of the technique, including ease of imidazole elution and significant purification in one step from protein expression systems [1]. Drawbacks to the method are few but include interference of tags with the quaternary structure and non-native random coil interfering with downstream techniques. To accommodate these issues, additional extensions or protease sites may be reengineered, but these introduce a nondesirable addition of random coil or a new experimental step in purification. The tag may be moved to the other terminus of the protein, but there are no further termini available if that one is also problematic. The presence of a half-dozen identified proteins in Escherichia coli with endogenous nickel affinity [2] suggests that metal affinity may be engineered on the surface of a protein instead of in a terminal tag. Logically, only two histidines might be required given that four of the six ligand positions on Ni2+ ions are commonly occupied by the solid phase ligands [1]. The optimal distance between a dyad pair of histidines can be extrapolated from inorganic structures and might be expected to be between 4 and 7 Å given the rotational range of motion around the CaACb bond in histidine. Tags are commonly engineered with six histidines on a terminus, however, and proteins with endogenous nickel affinity have between 3 and 6 histidines in a cluster, potentially providing a number of vicinal ligand pairs. Delineating the parameters of ⇑ Corresponding author. Fax: +1 509 313 5804. E-mail address: [email protected] (K.M. Hoffmann).
histidine location on the surface of proteins required to bind metal would define a broadly applicable extension of the metal affinity technique, expanding the number of possible metal affinity sites, minimizing the addition of random coil and non-native residues, and eliminating the need to remove affinity tags after purification. In addition, surface metal affinity could be used for reporter labeling or crystallography phasing. Before a general method can be developed, however, the characteristics present in proteins with endogenous metal affinity must be successfully applied to a similar system and the concept must be proven. Carbonic anhydrases (carbonate hydrolyase, EC 4.2.1.1) are zinc metalloenzymes that catalyze the reversible interconversion of CO2 and bicarbonate:
CO2 þ H2 O¡HCO3 þ Hþ :
ð1Þ
E. coli carbonic anhydrase (ECCA)1 is a member of the b-form of carbonic anhydrases, widely distributed in bacteria, yeast, and plant chloroplasts, serendipitously identified when it is bound persistently to a nickel column without any engineered tag [3]. Although all forms of carbonic anhydrases have convergently evolved to use metal ions in their catalytic mechanism, none uses nickel [4]. ECCA nevertheless binds so tightly to an Ni2+ column that 250 to 300 mM imidazole is needed to elute it off of the metal sites, comparable to the amount 1 Abbreviations used: ECCA, Escherichia coli carbonic anhydrase; ICP–OES, inductively coupled plasma–optical emission spectroscopy; HICA, Haemophilus influenzae carbonic anhydrase; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
http://dx.doi.org/10.1016/j.ab.2014.04.020 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
62 63 64 65 66 67 68 69 70 71 72 73 74
75 77 78 79 80 81 82 83 84 85
YABIO 11717
No. of Pages 3, Model 5G
15 May 2014 2 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151
Notes & Tips / K.M. Hoffmann et al. / Anal. Biochem. xxx (2014) xxx–xxx
needed to compete off a histidine-tagged protein [3]. There is no loss of ECCA activity using this purification method, and no nickel ions are retained in the protein itself based on inductively coupled plasma– optical emission spectroscopy (ICP–OES) measurements [3]. ECCA, like other b-carbonic anhydrases, has a dimer of dimers quaternary structure. The tetramerization interface between dimers is partly solvent exposed and contains two symmetry-related clusters of histidines. Each cluster includes six histidines from two chains (His72, His122, and His160 from one chain; His720 , His1220 , and His1600 from the other chain). The histidines are between 5 and 7 Å apart in the same chain, although the interchain distance between His72 and His1220 is closer at between 3.5 and 4.5 Å. Because nickel(II)–histidine coordination distances have been observed in protein structures at 1.8 to 2.4 Å [5], these side chains were hypothesized to be within the sphere of rotation necessary to optimize nickel coordination (see Fig. S1 in online Supplementary material) [2]. Haemophilus influenzae carbonic anhydrase (HICA), although a bcarbonic anhydrase family member sharing 38% identity, quaternary structure, and activity profile similar to ECCA, does not share the endogenous surface metal affinity and only partially conserves the cluster of histidines in the tetramerization interface (His72, His122, and Arg160), even though the residues overlay well. Mutating Arg160 to histidine in HICA sufficiently recreated ECCA’s metal affinity and allowed purification under similar conditions. Optimizing Arg160His HICA’s purification to minimize secondary retention during the binding and wash steps meant that the protein variant eluted earlier in the gradient but similarly achieved greater than 90% purity. Steady-state kinetic activity and oligomerization state were similar to wild-type HICA, establishing that the surface mutations did not influence the overall structure or function of the protein. This proof-of-concept system is the first step toward establishing the parameters for a general engineered surface metal affinity method. Arg160His was constructed using megaprimer PCR [6] with Pfu Ultra (Stratagene) polymerase and commercial oligonucleotides (Integrated DNA Technologies). A mutant oligonucleotide (50 Q1 CAATCTAGGGCACACATCAATTGT-30 ) was paired with the 50 terminal oligonucleotide primer PHI2X (50 -TGCCTGCAGTTATTATGTAT Q2 TTTCAAGATG-30 ) in the first polymerase chain reaction (PCR) to give a 226-bp product. This PCR product was used as a megaprimer in a second PCR with the 30 oligonucleotide primer PHI1QC (50 TGCCCATGGATAAAATTAAACAACTCTTT-30 ) to create the final mutated HICA gene. The final PCR product was used as a megaprimer in a QuikChange protocol. The Arg160His HICA gene was sequenced (Clemson University Sequencing Institute) to verify the introduction of the correct mutations. Expression of variant HICA proteins was as described previously [7]. Purification of ECCA and HICA proteins was as described for the wild-type ECCA protein except that detergent was omitted and 20 mM imidazole was used in the wash step for Arg160His HICA. Crude homogenates of overexpressed protein were bound to a nickel column (HisTrap HP, GE Healthcare) in buffer A (20 mM Tris–HCl [pH 8.0], 0.1 M NaCl, and 10 mM imidazole), washed, and purified to homogeneity using an AKTA FPLC (fast protein liquid chromatography) system (GE Healthcare) and a linear gradient of 20 to 500 mM imidazole. Fractions were pooled and desalted using a HiPrep 26/10 desalting column (GE Healthcare) and 20 mM Tris–HCl (pH 8.0) and 0.1 M NaCl buffer. Native oligomerization state was confirmed using the same buffer and a Sephadex 200 30/600 gel exclusion chromatography column (GE Healthcare). All proteins were quantified by absorbance at 280 nm [8], monomeric size was confirmed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE), and purity was estimated by densitometry analysis using the program ImageJ [9]. To explore whether the kinetic activity of Arg160His HICA was influenced by the mutation, steady-state kinetics experiments at
pH 9.0 were performed for both the HICA variant and wild-type ECCA, as described previously. Saturated solutions of CO2 were prepared by bubbling CO2 gas into water in a vessel maintained at 25.0 ± 0.1 °C, and dilutions were prepared by coupling two gas-tight syringes as described by Khalifah [10]. CO2 concentrations were calculated based on a 33.8-mM saturated solution at 25.0 °C [11]. All steady-state kinetic measurements were made at 25.0 °C using a Hi-Tech SF-61DX2 stopped-flow spectrophotometer and were carried out in the presence of 250 mM Na2SO4 for maximum enzyme stability and activity in dilute solution. Initial rates of CO2 hydration were measured using the changing pH indicator method described
Fig.1. (A) Normalized chromatography of Arg160His HICA and controls. 0 volume is after a 5-column-volume wash with 10 mM (ECCA and HICA) or 20 mM (Arg160His HICA) imidazole concentration and indicates the start of the gradient. ECCA (green dashed line) elutes at 11.2 ml and 278 mM imidazole. Wild-type HICA (red solid line) shows no retention on the nickel column. The increasing baseline is likely due to contaminants in the imidazole salt. Arg160His HICA (blue solid line) elutes at 7.7 ml and 190 mM imidazole. The linear imidazole gradient is shown as a black dotted line with the axis plotted on the right and does not reflect differing concentrations of imidazole in the wash step. (B) Representative gel exclusion chromatography elution with the x axis reflecting the retention volume (below the axis) and the retention volume of the standards used to estimate the molecular mass of the oligomer. Arg160His HICA elutes at a volume consistent with the wildtype biological tetramer. (C) SDS–PAGE gel of Arg160His HICA purification steps. Lane 1: soluble lysate from overexpression; lane 2: a fraction immediately preceding the main elution peak; lane 3: a fraction from the main elution peak with a distinct band at approximately 26 kDa; lane 4: from the shoulder peak at approximately 278 kDa; lane 5: pooled gel exclusion peak with a main monomer band at 26 kDa. (For interpretation of the references to color in this figure legend and the text, the reader is referred to the Web version of this article.)
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
152 153 154 155 156 157 158 159 160 161 162
YABIO 11717
No. of Pages 3, Model 5G
15 May 2014 3
Notes & Tips / K.M. Hoffmann et al. / Anal. Biochem. xxx (2014) xxx–xxx Table 1 Chromatography retention of Arg160His HICA as well as positive (ECCA) and negative (HICA) controls. Protein
Retention volume peak (ml)
[Imidazole] peak (mM)
Purity (%)
kcat (ms1)
kcat/Km (lM1 s1)
ECCA HICA Arg160His HICA
11.2 0 7.7
278 0 190
92.2 0 90.31
77 ± 7 69 ± 29a 37 ± 3
2.4 ± 0.7 4.3 ± 0.8a 2.7 ± 0.2
Note. Retention volume is calculated relative to the imidazole gradient on a 5-ml column, and a retention volume of 0 (zero) indicates that the lysate or protein was not on the column when the gradient was initiated. Purity was estimated from densitometry of SDS–PAGE gels using the program ImageJ [9]. Kinetic values of kcat and kcat/Km for HICA wild-type and variants were determined at pH 9.0 in reaction conditions of 40 mM 1,2-dimethylimidazole, 58 lM m-cresol purple, 1 lM ethylenediaminetetraacetic acid (EDTA), and 250 mM sodium sulfate. Reaction conditions for ECCA at pH 9.0 were 40 mM bicine, 25 lM m-cresol purple, 2.5 mM EDTA, and 250 mM sodium sulfate. a Wild-type HICA kinetic constants were originally reported by Cronk and coworkers [7]. 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
previously [10–12]. Values of kcat (reported on a per subunit basis) and Km were determined by nonlinear least squares fits to v/[E] versus [CO2] data using Origin 7.0 (Microcal). We have successfully engineered surface histidines for the nickel purification of HICA using an Arg160His mutation to complete a cluster of histidine residues (His72, His1220 , and His160) in ECCA that was partially conserved in HICA. However, the core of the cluster, His122 and His72 (and their symmetry mates), did not have metal affinity on their own (Fig. 1A, wild-type HICA control). Previous crystallization of HICA variants has revealed solvent phosphate molecules coordinated by several residues in the tetramerization interface, including Arg160 [13], suggesting that that the 160 position is a key solvent-exposed histidine. The purification of Arg160His HICA based on the previously published ECCA purification buffers eluted the main peak at a similar concentration to ECCA (278 mM at the peak; Fig. 1A, green line) but had significant contamination when washed with a 10-mM imidazole concentration (data not shown). Wild-type HICA protein had no affinity for the nickel column (Fig. 1A, red line). To optimize the purification of Arg160His HICA, we increased the initial imidazole concentration to 20 mM and observed the elution of a specifically bound protein at 190 mM in the gradient (Fig. 1A, blue line). Using the optimized purifications, we purified Arg160His HICA to Q3 greater than 90% purity with a single step (Fig. 1C and Table 1). To confirm that the Arg160His mutation did not alter the native chemistry of HICA, we performed steady-state kinetics and found that kcat = 37 ± 3 ms1 and kcat/Km = 2.7 ± 0.2 lM1 s1 (Table 1). These values are similar to wild-type numbers and well within the range of nonconsequential HICA variant activity observed preQ3 viously. Because the Arg160His mutation was located in the oligomerization interface, we confirmed that Arg160His HICA eluted at a size consistent with a tetramer (122 ± 24 kDa) using gel exclusion chromatography (Fig. 1B). These experiments establish that the location and number of mutations does not disrupt the native structure or behavior of the protein. Using published crystal structure PDB 2A8D, we have modeled the Arg160His mutation (Fig. S1) and examined the ability of the residues to make the necessary ligand distances to a metal as well as the solvent accessibility of the cluster. Rotation of the nearby His72 and His1220 about the CaACb bond easily allowed His72 and Arg160His to rotate to within 3.3 Å of each other, sufficient to simultaneously accommodate approximately 2-Å ligand distances to a nickel ion with reasonable angles and good solvent exposure. The location of Arg160His on the edges of the buried histidine clusters means that there are four possible dyad nickel binding sites per HICA tetramer. Whether the metal affinity is due exclusively to multiple dyad histidine pairs or whether the rest of the cluster plays activating roles is not clear, but either hypothesis would be consistent with most other identified proteins with endogenous nickel affinity presenting clusters of at least three exposed proximal histidines [2]. Compared with the published three-column purification scheme (cation exchange, hydrophobic exchange, and gel exclusion chromatography) for HICA [9], metal affinity purification is a substantial improvement in time and resources. Beyond the advan-
tage for the carbonic anhydrases, however, this method is a proofof-concept that may be extended to numerous other systems in which a terminal histidine tag is undesirable such as for structural studies requiring a native-like tertiary structure. Vicinal histidines or designed surface clusters also have the extended potential to be used for reporter labeling or deliberate metal ion binding to a protein’s surface. We have shown that histidine-based metal affinity may be engineered onto the surface of a protein rather than restricting an engineered tag to one of two termini. To extend this work to a general method, we will repeat the histidine mutations in a more completely surface-exposed area of the protein and establish the minimal histidine character needed for nickel affinity as well as the stoichiometry of metal binding via ICP–OES. Future work will also explore the role of extra histidines beyond the minimum dyad pair to create optimal surface metal affinity.
217
Acknowledgments
232
The authors gratefully acknowledge the helpful contributions of Roger S. Rowlett and also thank Jeffrey Cronk, Hannah Maul, Dana Walters, and the students of Chem 443L for their assistance.
233
Appendix A. Supplementary data
236
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.020.
237
References
239
[1] J. Crowe, B.S. Masone, J. Ribbe, One-step purification of recombinant proteins with the 6His tag and Ni-NTA resin, Mol. Biotechnol. 4 (1995) 247–258. [2] V.M. Bolanos-Garcia, O.R. Davies, Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography, Biochim. Biophys. Acta 1760 (2006) 1304–1313. [3] J.D. Cronk, J.W. O’Neill, M.R. Cronk, J.A. Endrizzi, K.Y.J. Zhang, Cloning, crystallization, and preliminary characterization of a -carbonic anhydrase from Escherichia coli, Acta Crystallogr. D 56 (2000) 1176–1179. [4] R.S. Rowlett, Structure and catalytic mechanism of the-carbonic anhydrases, Biochim. Biophys. Acta 2010 (1804) 362–373. [5] R. Balakrishnan, N. Ramasubbu, K.I. Varughese, R. Parthasarathy, Crystal structures of the copper and nickel complexes of RNase A: metal-induced interprotein interactions and identification of a novel copper binding motif, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 9620–9625. [6] G. Sarkar, S.S. Sommer, The ‘‘megaprimer’’ method of site-directed mutagenesis, BioTechniques 8 (1990) 404–407. [7] J.D. Cronk, R.S. Rowlett, K.Y.J. Zhang, C.K. Tu, J.A. Endrizzi, J. Lee, P.C. Gareiss, J.R. Preiss, Identification of a novel noncatalytic bicarbonate binding site in eubacterial-carbonic anhydrase, Biochemistry 45 (2006) 4351–4361. [8] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem. 182 (1989) 319–326. [9] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9 (2012) 671–675. [10] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase: I. Stop-flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971) 2561–2573. [11] A.F. Ghannam, W. Tsen, R.S. Rowlett, Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2, J. Biol. Chem. 261 (1986) 1164–1169. [12] R.S. Rowlett, N.J. Gargiulo, F.A. Santoli, J.M. Jackson, A.H. Corbett, Activation and inhibition of bovine carbonic anhydrase III by dianions, J. Biol. Chem. 266 (1991) 933–941. [13] R. Rowlett, C. Tu, J. Lee, A. Herman, Allosteric site variants of Haemophilus influenzae b-carbonic anhydrase, Biochemistry 48 (2009) 6146–6156.
240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
218 219 220 221 222 223 224 225 226 227 228 229 230 231
234 235
238
273
Please cite this article in press as: K.M. Hoffmann et al., Surface histidine mutations for the metal affinity purification of a b-carbonic anhydrase, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.04.020
