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Efficient and Flexible Preparation of Biosynthetic Microperoxidases Erin C. Kleingardner, Wesley B Asher, and Kara L. Bren Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00915 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Biochemistry

Efficient and Flexible Preparation of Biosynthetic Microperoxidases Erin C. Kleingardner, Wesley B. Asher, and Kara L. Bren*

Department of Chemistry, University of Rochester, Rochester, New York 14627-0216 USA

*To whom correspondence should be addressed. [email protected]

Funding This work is supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy, Grant No. DE-FG0209ER16121 (expression method development by E. C. K.) and by the National Science Foundation grant CHE-1409929 (characterization and analysis by W. B. A. and K. L. B.) This work also was supported by an Elon Huntington Hooker Fellowship to W. B. A.

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Abbreviations: Ccm: Cytochrome c maturation; CD: circular dichroism; CPD: cysteine protease domain of the Vibrio cholerae MARTX toxin protein; cyt c: cytochrome c; HIS: histidine-immobilized Sepharose; InsP6: inositol hexakisphosphate; IPTG: isopropyl β-D-1-thiogalactopyranoside; LB: Luria-Bertani; MARTX: Multifunctional autoprocessing repeats-in-toxin; MP: microperoxidase; NaPi: sodium phosphate; Ni-NTA: Ni(II)-loaded nitrilotriacetic acid agarose; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; YT: yeast extract and tryptone

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Abstract Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin, and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as Nterminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., Bren, K. L. (2010) Protein Sci. 19, 1830-1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.

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Microperoxidases (MPs) are biosynthetic heme-peptide conjugates that have been widely used as models for heme protein active sites1-6 and for investigations of mechanisms of peroxidase activity.7,

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MPs and their derivatives also have been used in energy conversion

applications as catalysts for hydrogen evolution,9 oxygen reduction,6, 10 and in fuel cells.11 MPs consist of a heme covalently attached to a peptide via thioether bonds to two Cys residues within a Cys-X-X-Cys-His (CXXCH) motif (Figure 1). The peptide that is attached to the heme in MPs donates an axial His ligand to the heme iron, confers water solubility on the porphyrin, and influences porphyrin structure.1 The peptide moiety also facilitates attachment of the metalloporphyrin to other structures such as electrodes,10-12 nanotubes,13, 14 and nanoparticles.15

Figure 1. Heme c with a Cys-Xaa-Xaa-Cys-His attachment motif. The “Xaa” residues may be any of the 20 canonical amino acids. MPs are usually prepared by proteolytic digestion of horse heart cytochrome c. For example, the eleven-residue MP-11 is prepared by pepsin digestion, and the eight-residue MP-8 is prepared by pepsin and trypsin digestion.16,

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MPs with different sequences have been

generated by proteolysis of other cytochromes c, including Shewanella oneidensis MtrA18 and Marinobacter hydrocarbonoclasticus cytochrome c552.19 Although cytochrome c digestion has successfully generated MPs with different peptide sequences, reliance on proteolytic cleavage of known cytochromes c limits the ability to vary peptide length and amino acid sequence as desired. Synthetic methods for producing covalent peptide-porphyrin conjugates allow greater flexibility,20 but this approach requires a highly substituted synthetic porphyrin, and the heme c attachment (Figure 1) has not been produced synthetically. Nevertheless, fully synthetic proteins and peptides with covalently attached metalloporphyrins have been successfully produced in

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amounts sufficient for structural and physical characterization, and represent a valuable complementary approach.21-23

Figure 2. Schematic of the cytochrome c maturation (heme attachment) process mediated by the Ccm system in E. coli.24, 25 Here, the maturation of a CXXCH-containing tag to a cysteine protease domain (CPD), an approach used in this work, is illustrated. Heme and the apoprotein are synthesized in the E. coli cytoplasm and both are translocated to the periplasm, where the protein’s signal sequence is cleaved. The heme is transported via a heme chaperone CcmE to CcmF, which facilitates heme attachment to apoprotein. Electron transfers facilitated by DsbD, CcmG, and CcmH reduce the Cys residues in the CXXCH motif. The structure of the CPD is from the protein data bank (3EEB).26 A major advance in heme peptide biosynthesis was made by Braun and Thöny-Meyer, who prepared recombinant MPs with peptide lengths between 11 and 26 residues by direct expression of His-tagged peptides from the N-terminal region of Bradyrhizobium japonicum cytochrome c550 containing the CXXCH heme-attachment motif in Eschericia coli.27 The CXXCH sequence is targeted for covalent heme attachment by the E. coli cytochrome c maturation (Ccm) proteins (schematic and summary given in Figure 2), and the resulting heme peptides have heme covalently attached to this motif. While it is a viable system for expressing MPs, preparation of detectable amounts of MP required the inclusion of a His-tag (C-terminal His6 sequence) sequence on the peptide, yielding a hexacoordinate heme site. Here, we present two novel methods for the biosynthesis of new MPs matured using the E. coli Ccm system, summarized in Figure 3. Notably, for both methods, MPs with an open

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coordination site at the heme are prepared at levels sufficient for further modification and study. Notably, the sequences chosen have no relationship to known cytochromes c, demonstrating flexibility in design. In one method (Figure 3b), heme peptides are expressed and matured as Cterminal heme-peptide tags (or "heme tags") on the protein azurin. Azurin was chosen because it can be overexpressed at high levels in E. coli and was previously shown to be amenable to tagging with a heme peptide.28 Following expression and maturation, the heme-tagged azurin is purified on histidine immobilized Sepharose (HIS) resin previously developed for purifying heme-tagged proteins,29 and then cleaved with the endopeptidase factor Xa to yield the heme peptide. In the second method (Figure 3a), heme peptides are expressed as N-terminal tags on Vibrio cholerae MARTX cysteine protease domain (CPD), using a system developed by Shen and Bogyo to facilitate the expression of soluble proteins.30 The protease activity of CPD is activated by inositol hexakisphosphate (InsP6), leading to autocleavage at a CPD N-terminal leucine residue to liberate the protein or peptide of interest. Here, we show that these methods facilitate the preparation of designed MPs of selected sequences (Table 1) and an open heme coordination site.

Table 1. Microperoxidase sequences. The CXXCH heme attachment sequences are underlined. Peptide Cleavage Sequence Molecular Yield methoda

Massb

(pmol/L)

MP-24

Xa

VEAFEKKVAAFESKCAACHAKSKR

3256

250

MP-13

Xa

GYASCWACHEEEE

2130

160

MP-24VDAL

CPD

VEAFEKKVAAFESKCAACHAKSKRVDAL 3654

300

MP-13VDAL

CPD

GYASCWACHEEEEVDAL

2529

80

MP-

CPD

GYASCAACHEEEEVDAL

2413

150

13VDAL/A6 a

Xa indicates cleavage from azurin using factor Xa. CPD indicates expression on CPD followed by autolysis. bCalculated for peptide sequence as shown with heme

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Figure 3. Schematic representation of two methods for preparation of MPs developed in this work. (A) Expression of an N-terminal heme tag to CPD followed by autocleavage. (B) Expression of a C-terminal heme tag to azurin followed by factor Xa cleavage. SS = signal sequence, CPD = cysteine protease domain, VDAL = autocleavage site for CPD (cleaves after L), His6 = His tag, IEGR = factor Xa cleavage site (cleaves after R). Peptides and proteins are shown with the N-terminus on the left and C-terminus on the right.

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Experimental Procedures Plasmid construction. Synthetic DNA and primers were prepared by Integrated DNA Technologies (Coralville, IA) and sequences are given in Tables S1 and S2. Preparation of the pETHmPep(Xa) plasmid (Figure S1) for expression of peptides on the C-terminus of azurin (denoted MP-24 and MP-13, see Table 1) was achieved by modification of the pETAzHm14 plasmid (ampicillin resistant), which contains the azurin structural gene including its signal sequence for excretion to the periplasm.28 The synthetic DNA sequence encodes the last four amino acids of azurin, a linker, the designed heme peptide sequence, and a factor Xa recognition site (Figure 3b). The MP-24 synthetic gene (Table S1) was ordered as a single-stranded ultramer and was amplified by PCR using MP-24fwd and MP-24bwd primers (Table S2). The amplified MP-24 DNA was cloned into the KpnI/SpeI site of pETAzHm14 (Figure S1). A plasmid to express MP-13 as a C-terminal tag to azurin was constructed in a similar manner: MP-13fwd and MP-13bwd primers were used to amplify the MP-13 artificial gene, which was cloned into pETAzHm14 as described for MP-24. Plasmids for the expression of heme peptides on the N-terminus of CPD (pETHmPep(CPD) (ampicillin resistant), Figure S1) were based on the pET-CPDSalI vector harboring the gene expressing the Vibrio cholerae MARTX toxin CPD domain,30 provided as a gift from Dr. Aimee Shen and Dr. Matthew Bogyo. This expression vector encodes an Nterminal pelB signal sequence for periplasmic excretion, and a C-terminal His-tag to the CPD domain. The MP-24VDAL artificial gene (Table S1) was amplified by PCR using primers MP24VDALfwd and MP-24VDALbwd, and the MP-13VDAL gene (Table S1) was amplified with its corresponding primers (Table S2). These products, encoding peptides to be expressed on the N-terminus of CPD, were cloned into the NcoI/SalI site of pET-CPDSalI, between the pelB and CPD sequences. A plasmid to express MP-13VDAL/A6, a variant of MP-13VDAL, was generated with the QuikChange (Stratagene) method, using mutagenic primers MP13VDAL/A6fwd and MP-13VDAL/A6bwd (Table S2).

Expression of Heme-tagged Proteins. Protein expression was carried out in BL21(DE3)Star E. coli (Invitrogen) transformed with the appropriate pETHmPep(Xa) or a pETHmPep(CPD) derivative as well as pEC86 (Cmr), which encodes the E. coli cytochrome c maturation genes ccmABCDEFGH.31 Expression of azurin with a C-terminal heme tag was

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modified from the method described previously.28 A 5-mL Luria-Burtani (LB) culture, shaken at 625 rpm at 37 ˚C for 8-9 hours was used to inoculate 2 L of LB medium, which was then grown anaerobically at 120 rpm and 37 ˚C. Cultures contained 50 mg/L ampicillin and 50 mg/L chloramphenicol. Once an OD600 of 0.6-0.8 was reached, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a concentration of 0.4 mM, and cells were harvested 6 hours after induction and stored at −20 ˚C. Expression of CPD with an N-terminal heme peptide tag was optimized from the method described by Bogyo, et al.32 A 5-mL 2x YT culture, grown overnight at 625 rpm and 37 ˚C, was used to inoculate 250 mL of 2x YT medium in a 1-L flask at a dilution of 1:500. Cultures contained 50 mg/L ampicillin and 50 mg/L chloramphenicol. Cultures were grown aerobically with shaking at 200 rpm for 24 hours at 25 ˚C, and cells were harvested by centrifugation at 10,000 × g. Cell pellets were resuspended in 30 mL of lysis buffer 1 (500 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, 10 % glycerol, pH 7.5), flash-frozen, and stored at −20 ˚C. Expression of heme-tagged CPD was found to be dependent on the size of the growth flask, with best results for the 1-L flask. Cell pellets were red in color, indicating overexpression of heme protein (Figure S2).

Purification of Microperoxidases. For purification of heme-tagged azurin, E. coli pellets were thawed, solubilized in lysis buffer 2 (50 mM Tris, 1 % Triton-X100, 4 mg/ml lysozyme, 1 U/mL DNAse, pH 7.6) and incubated at 30 ˚C for 1 hour. Following centrifugation at 10,000 × g, the supernatant was adjusted to a pH of 8.8 with NaOH and then purified by anion-exchange chromatography on a diethylaminoethyl cellulose column equilibrated with 20 mM Tris, pH 8.8. Protein was eluted with 20 mM Tris/150 mM NaCl, pH 8.8. The partially clarified lysate was exchanged into 50 mM sodium phosphate (NaPi), pH 7, and then affinity purified on HIS resin28 in a 2 × 8 cm column equilibrated with 50 mM NaPi, pH 7. The resin was washed with 2 column volumes of 50 mM NaPi, pH 7 and heme-tagged azurin constructs were eluted in 50 mM NaPi/300 mM imidazole, pH 7. To remove imidazole, the sample pH was adjusted to 4.5, and the buffer was exchanged using a PD-10 size-exclusion column into 50 mM NaPi, pH 4.5. Heme tag cleavage was performed in 20 mM Tris/50 mM NaCl, pH 6.5, with a ratio of 3 nmol heme-tagged azurin per 1 mg factor Xa. Heme peptides were purified on HIS28 resin in a 2 × 8 cm column equilibrated with 50 mM NaPi, pH 7.0. The column was washed with 2 column volumes of 50 mM NaPi/pH 7.0, and heme peptides were eluted in 50 mM NaPi, 300

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mM imidazole, pH 7.0. To remove imidazole, the pH was adjusted to 4.5, and the buffer was exchanged into 50 mM NaPi, pH 4.5 on a PD-10 size-exclusion column. For the preparation of heme peptides from CPD domains, CPD-heme peptide constructs were purified using Ni2+-NTA affinity chromatography. Cells were thawed and lysed by sonication on a Branson Sonifier 450 in lysis buffer 1. Cells were harvested by centrifugation at 15,000 × g for 30 minutes at 4 ˚C. For batch adsorption, 30 mL of lysate was bound to 2.5 mL of Ni2+-NTA beads by shaking at 150 rpm at 25 ˚C for 2 hours. The Ni2+-NTA resin with adsorbed protein was washed three times by pelleting at 1,500 × g, decanting, and resuspending the beads in 10 mL of lysis buffer 1. Finally, the Ni2+-NTA beads were pelleted, packed in a 2 × 8 cm column, and washed with 20 mL of lysis buffer 1. To cleave heme peptide from the CPD domain, 5 mL of cleavage buffer (lysis buffer 1 with 200 µM InsP6) was added to the Ni2+-NTA beads to make a slurry. This was shaken at 200 rpm and 25 ˚C for 1 hour. The supernatant, containing the cleaved heme peptide, was collected, and beads were washed with lysis buffer 1 to remove additional cleaved peptide. The column was washed with 50 mM NaPi/300 mM imidazole, pH 7 to remove bound CPD. Heme peptides were then exchanged into 50 mM NaPi, pH 7.0 buffer using a PD-10 column.

Characterization of Microperoxidases. Tris-Tricene SDS-PAGE peptide gels (BioRad) were loaded with purified products and run at 100 V for 1 hour. Heme staining of the gel was carried out as described.33 UV-vis absorption spectra were collected on a Shimadzu UV-2401PC spectrometer using a 1-cm path length quartz cuvette. The pyridine hemochrome method for determining extinction coefficients was used as described.34 Circular dichroism (CD) spectra were collected on an Aviv Instruments model 202 spectropolarimeter on 100 mM heme peptide samples in a 0.100-cm path length quartz cuvette. MALDI-TOF mass spectrometry was carried out on a Bruker Autoflex III mass spectrometer using an α-cyano-5-hydroxycinnamic acid matrix.

Results Construct Design. Two unrelated peptides were designed for this proof-of-principle study, MP-24 and MP-13 (Table 1). The MP-24 24-mer peptide sequence was based on the amphipathic nano-1 peptide that forms α-helices when bound to hydrophobic moieties such as

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carbon nanotubes.35 The MP-24 sequence includes two repeating heptads with nonpolar residues placed on one face of a predicted alpha helix, followed by a CAACH sequence for covalent heme attachment. A sequence of five polar or charged residues follows the CAACH to enhance solubility. The 13-mer MP-13 sequence places a CWACH heme-attachment sequence between short polar sequences. A Trp was placed within the heme attachment motif to test whether the maturation process can accommodate the presence of a bulky residue at this position. The derivatives of these peptides prepared from the CPD construct contain an additional four residues (VDAL) at the C-terminus because CPD cleaves after the Leu residue of this four amino acid sequence. In addition to these constructs, the Trp in MP-13VDAL was mutated to Ala for the CPD-linked heme peptide to test effects on yield.

Preparation of Microperoxidases. Preparation of heme peptides as tags fused to azurin involved four steps: 1) anion-exchange and 2) HIS affinity purification of azurin-heme peptide from cell lysate, 3) factor Xa cleavage of the heme peptide from the C-terminus of azurin, and 4) purification of the cleaved heme peptide by HIS chromatography. Two of the HIS column purification steps are shown in Figure S3. Preparation of heme peptides using CPD as a carrier was performed by a three-step procedure: 1) batch purification of CPD-heme peptide fusions on a Ni2+-NTA column, 2) application of InsP6 to the column followed by elution of cleaved heme peptide and 3) HIS chromatography to purify the heme peptide. Peptide yields were measured using UV-vis spectroscopy (see below). Typical yields of purified heme peptide (Table 1) range from ~200 µg to 800 µg/L of culture (80 pmol/L to 300 pmol/L), which provides ample material for further use and characterization. Notably, the direct expression method for hexacoordinate MPs provided a maximum yield of (202 µg/L, or 58 pmol/L),27 indicating that expression as a heme tag followed by cleavage provides a competitive yield of MPs. Furthermore, it is notable that the MPs prepared here have an open coordination site, while similar derivatives could not be prepared by direct expression and maturation of the peptides.

Characterization of Microperoxidases. Using the pyridine hemochrome method, the extinction coefficient at the Soret band maximum at pH 7.0 was found to be 117 mM-1 cm-1 for MP-24VDAL and 113 mM-1 cm-1 for MP-13VDAL and 117 mM-1 cm-1 for MP-13VDAL/A6 (using variants prepared from CPD cleavage) at pH 7.0, and these values were used in

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quantitation. SDS-PAGE gels visualized by heme staining confirmed the isolation of peptides with covalently attached heme of the expected molecular masses (Figure S4, see Table 1 for predicted masses). Faint bands consistent with uncleaved product can be seen for MP-13VDAL and MP-13. To determine whether the heme peptides had the expected composition, MALDITOF mass spectrometry was carried out, with results summarized in Table S3. All heme peptides were found to have a mass within one unit of the predicted mass. However, for preparation of the heme peptides using the CPD fusion protein, two different products of signal sequence cleavage resulted: one with the target sequence, and one with an additional residue (Met). UV-vis spectra are typical of ferric heme peptides, with Soret maxima (pH 7.0) ~402 nm (Figure 4; value is dependent on pH and solvent). The MP-13 peptides have higher intensity in their spectra in the near UV region, consistent with the presence of a Trp and Tyr residue. Similarly, MP-13VDAL/A6, which has a Tyr residue, has greater intensity in the near-UV relative to MP-24VDAL. MPs tend to dimerize with the N-terminus of one peptide coordinating to the open site on the heme on another peptide, yielding low-spin heme at sufficiently high concentrations, although the presence of a small band ~620 nm indicates some high-spin character. Using methanol as a solvent decreases aggregation,36 and the resulting UV-vis spectrum is typical of a high-spin ferric heme (Figure S5). Acetylation of microperoxidases is another approach to preventing dimerization that facilitates the use of these compounds in water.17, 37

Figure 4. UV-vis spectra of MP-24VDAL (orange), MP-13VDAL (cyan) and MP13VDAL/A6 (dark green). Samples are in 50 mM NaPi, pH 7.0.

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To determine if MP-24 has α-helical character in solution as predicted,35 we collected a CD spectrum in a 50:50 methanol:water mixture. Methanol was added to inhibit aggregation.36 The CD spectrum of MP-24VDAL exhibits minima at 205 and 222 nm, consistent with the presence of some α-helical structure (Figure S6).

Discussion Here, we present two new approaches to the preparation of novel MPs. Previously reported methods rely on proteolysis of cytochromes c38 or the expression and maturation of small peptides.27 The approaches presented here are based on expressing and maturing heme peptides as cleavable tags to carrier proteins, allowing the dialing in of a desired sequence and providing yields of pentacoordinate heme peptide products sufficient for further study and use. These two methods yield similar results to each other in terms of MP yield (Table 1) and purity (Figure S4) for the test peptides used. The advantage of the CPD-based approach is that the CPD carrier protein undergoes autocleavage to liberate the MP, decreasing materials cost and facilitating the cleavage step. However, the CPD-derived heme peptides necessarily include a VDAL sequence on their C-termini as a result of the CPD cleavage, which for some applications may not be desirable. The approach using azurin as a carrier protein requires use of an endopeptidase, but the resulting cleaved MPs contain only the desired amino acids in their sequences. This approach could be modified to use any desired carrier protein that is amenable to excretion to the periplasm.28 These results are in accord with other reports that the E. coli Ccm apparatus is capable of maturing a wide range of peptide sequences that contain a CXXCH motif.27,

39-41

Factors

determining heme attachment efficiency, however, remain unclear. The successful maturation of both six-coordinate (His-tagged) peptides and five-coordinate heme tags to proteins indicates that protection of the target structure from degradation is sufficient to yield a product. Furthermore, we find that the maturation of a peptide tag containing a bulky residue (CWACH) results in lower yields (by ~50%) than maturation of the sequence substituted with an Ala (CAACH; Table 1). Further analysis of such effects will benefit our understanding of the Ccm system and how to exploit it to prepare novel structures.

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Although MPs have been known and studied for decades, there remains significant interest in their use as model systems, catalysts, and sensors. Substituting the heme iron with other metals is readily done to alter reactivity, but altering the nature of the attached peptide to impact properties has been relatively unexplored. For example, changing the heme attachment sequence is expected to affect heme conformation (nonplanarity),42 which influences electronic structure and reactivity.43-50 Furthermore, altering the peptide sequence will affect solubility, overall charge, and affinity for various resins, electrodes, or other surfaces. These expression approaches open up the possibility to explore these variations and applications.

Acknowledgements The authors thank Prof. Linda Thöny-Meyer for the gift of pEC86 and Prof. Yi Lu for providing the azurin gene.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications Website (http://pubs.acs.org) at DOI: 10.1021/acs.biochem.xxxxxxx Synthetic DNA and primer sequences Mass spectrometry results Plasmid maps Images of cell pellet, purification process, and gel electrophoresis results UV-vis and CD spectra

References

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(1) Ma, J. G., Laberge, M., Song, X. Z., Jentzen, W., Jia, S. L., Zhang, J., Vanderkooi, J. M., and Shelnutt, J. A. (1998) Protein-induced changes in nonplanarity of the porphyrin in nickel cytochrome c probed by resonance Raman spectroscopy. Biochemistry 37, 5118-5128. (2) Tezcan, F. A., Winkler, J. R., and Gray, H. B. (1998) Effects of ligation and folding on reduction potentials of heme proteins. J. Am. Chem. Soc. 120, 13383-13388. (3) Cowley, A. B., Lukat-Rodgers, G. S., Rodgers, K. R., and Benson, D. R. (2004) A possible role for the covalent heme-protein linkage in cytochrome c revealed via comparison of N-acetylmicroperoxidase-8

and

a

synthetic,

monohistidine-coordinated

heme

peptide.

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