A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli—Expression and characterization of cytochrometagged Complex I subunits
Tobias Gustavsson, Maria Trane, Vamsi K. Moparthi, Egle Miklovyte, Lavanya Moparthi, Kamil Go´recki, Thom Leiding, Sindra Peterson A˚rsko¨ld, and Cecilia Ha¨gerha¨ll* Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, 22100 Lund, Sweden Received 20 March 2010; Revised 2 May 2010; Accepted 10 May 2010 DOI: 10.1002/pro.424 Published online 27 May 2010 proteinscience.org
Abstract: Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the Cterminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an Em of about 1170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. Abbreviations: Ampr, ampicillin resistance; BSA, bovine serum albumin; BTP, bis-tris-propane; Cmr, chloramphenicol resistance; IPTG, isopropyl b-D-1-thiogalactopyranoside; LB, Luria Bertoni; LHON, Leber’s hereditary optic neuropathy; MrpAcyt, MrpA with a fused cytochrome c tag; MrpDcyt, MrpD with a fused cytochrome c tag; NuoHcyt, NuoH with a fused cytochrome c tag; NuoLcyt, NuoL with a fused cytochrome c tag; NuoMcyt, NuoM with a fused cytochrome c tag; NuoNcyt, NuoN with a fused cytochrome c tag; PCR, polymerase chain reaction; PVDF, polyvinylidene fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Grant sponsors: Carl Tryggers Foundation, Crafoord Foundation, Research School in Pharmaceutical Science, FLA¨K, The Sven and Lilly Lawsky Foundation. *Correspondence to: Cecilia Ha¨gerha¨ll, Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Box 124, 22100 Lund, Sweden. E-mail:
[email protected]
C 2010 The Protein Society Published by Wiley-Blackwell. V
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Keywords: cytochrome c; fusion proteins; covalently bound heme; NADH:quinone oxidoreductase; NuoH; NuoL; NuoM; NuoN; MrpA; MrpD; Escherichia coli; Bacillus subtilis Introduction The most convenient way to express a prokaryote membrane protein is in Escherichia coli, where many suitable strains and vectors are available.1 However, there are many technical obstacles to overcome to obtain good yields of membrane proteins when compared with soluble proteins, the need for posttranslational translocation through the Sec-machinery,2,3 and the limited physical space in the membrane being just the beginning. Many membrane proteins are also poorly antigenic, and most also stain poorly, making protein expression cumbersome to monitor and optimize. The proton-pumping NADH:ubiquinone oxidoreductase (Complex I) of prokaryotes has a molecular mass of about 500 kDa, where the NuoBCDEFGI domain constitutes the hydrophilic arm harboring all known prosthetic groups, and the NuoAHJKLMN domain constitutes the hydrophobic arm, embedded in the membrane. The structure of the hydrophilic arm from the bacteria Thermus thermophilus has ˚ resolution,4,5 revealing the locabeen solved at 3.3 A tion of the flavin mononucleotide and nine iron-sulfur clusters. The membrane-spanning domain of Complex I harbors important components of the energy-conserving machinery, but remains poorly understood compared with the protruding, watersoluble part. Two-dimensional crystals of the mem˚ rebrane domain resulted in images with about 8 A 6 solution, but no high-resolution structure exists. The structural features of the membrane-spanning part of the Complex I enzyme have been mapped by experimentally supported transmembrane topology models,7–10 crosslinking,11 and fractionation studies,12 combined with single particle analyses.13 We and others have tried to express the individual membrane-spanning Complex I polypeptides in E. coli using a variety of strains and state-of-the-art strategies, his-tagged proteins, etc., without much success. Antiporters of the Mrp type (also denoted Sha, Mnh, or Pha) from different organisms have been expressed in E. coli strains deleted of their native antiporters, but mainly for functional complementation studies14–19 and not for larger scale protein production. The homologous Complex I subunits NuoL, NuoM, and NuoN have never been overexpressed in E. coli in any appreciable amounts. Only a truncated form of NuoL was successfully produced from a plasmid-encoded gene.20 The whole NuoL, fused with a protein A domain, has thereafter been produced in yeast endoplasmatic reticulum with seemingly retained functionality.21 Other studies of the NuoL, NuoM, or NuoN subunit function have been performed by modifying the chromosomally
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located genes.22–25 Here, we present protein constructs where the C-terminal end of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli have been genetically fused to a cytochrome c domain (Fig. 1). The covalently bound heme in cytochrome c renders the proteins visible by optical spectroscopy and can be used to monitor, quantify, and orient the polypeptides in a plethora of novel future experiments. Surprisingly, the cytochrome c fusion proteins could also be produced with much higher yields compared with all previous attempts to produce the proteins without the fused cytochrome c. We suggest that the compact cytochrome c domain protects the C-terminal of these polypeptides from proteolysis during growth.
The choice of cytochrome c The Bacillus subtilis cytochrome c550 is a 13-kDa protein, encoded by the cccA gene, displaying a Q-band absorption peak at 550 nm. This cytochrome c contains an N-terminal transmembrane helix anchoring it to the membrane, as is common in Gram-positive organisms.26 The heme is covalently bound by a classical c-type CXXCH motif, where the two cysteines form covalent thioether bonds to the heme group. The histidine in the motif and a C-terminally located conserved methionine serve as axial ligands to the heme iron. Cytochrome c550 has a redox midpoint potential of 178 mV.27 The presence of a membrane anchor in the form of the N-terminal transmembrane helix made this cytochrome a particularly suitable candidate for creating a cytochrome-containing fusion protein, as the membrane anchor helix could be replaced by a transmembrane helix from the target protein, without any anticipated structural modifications. Furthermore, large amounts of holo-cytochrome c550 have previously been produced in E. coli under aerobic or microaerophile conditions.28 In bacteria, the cytochrome c-type covalent thioether bonds can only form in the oxidizing environment on the periplasmic side of the membrane. Thus, only proteins where the C-terminal end is located in the periplasm can be tagged with holo-cytochrome c. On the other hand, once the heme is incorporated, it will not be lost from the protein during purification or handling and will be retained even under the denaturing conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The choice of the membrane-spanning Nuo proteins The membrane-spanning domain of Complex I is bound to harbor important components of the energy-coupling machinery. The NuoH subunit
Cytochrome c Fusion Proteins for Production of Membrane Proteins
Figure 1. Schematic drawing of the fusion proteins is presented in this article, where the last transmembrane helix of the Nuo protein replaces the natural membrane anchor helix in B. subtilis cytochrome c550. A model of the cytochrome c550 is shown in A. The topology of the NuoH protein, shown in B, is based on Roth and Ha¨gerha¨ll,7 and the topology of the antiporter-like proteins NuoL (shown in C) and NuoM/NuoN/MrpD (shown in D) is based on Mathiesen and Ha¨gerha¨ll.8 NuoM, NuoN, and MrpD have the same transmembrane topology and number of transmembrane helices, thus only one model protein is shown representing all three proteins. MrpA is similar to NuoL, but contains an additional C-terminal extension (not shown).
[Fig. 1(B)] is located in the membrane-spanning domain of Complex I and has long been a strong candidate for harboring a quinone binding site. The NuoH subunit (denoted ND1 in mitochondria) has been specifically photolabeled with a rotenone analog.29 A number of mitochondrial mutations correlating with the inherited disease Leber’s hereditary optic neuropathy (LHON) were found in the NuoH subunit.30 Mutations mimicking some of the LHON mutants were introduced in NuoH (the equivalent of NQO8) of Paracoccus denitrificans and shown to affect quinone reductase activity.31 If the subunit could be overexpressed and purified without the rest of the enzyme complex, the putative quinone binding site could be probed by direct azidoquinone labeling and mass spectrometry. The C-terminal end of NuoH is located in the periplasm,7 making it a candidate for cytochrome c tagging (Fig. 1). As mentioned before, the antiporter-like subunits NuoL, NuoM, and NuoN [Fig. 1(C,D)] are all homologous to the antiporters of the Mrp/Sha/Mnh/ Pha type, making them strong candidates for containing proton and/or ion channels in Complex I. An
Gustavsson et al.
unresolved controversy exists over whether Complex I translocates protons exclusively or sodium as well. The proton/electron stoichiometry of Complex I has been scrutinized in many labs over the years (summarized in32), reaching a consensus of 4Hþ/2e in Complex I in tightly coupled submitochondrial particles from bovine heart. At the same time, it was demonstrated that Complex I from Klebsiella pneumonie translocated Naþ,33 and other experiments suggested that Complex I from E. coli34 also functions as a primary sodium pump. It is not known whether the NuoL, NuoM, and NuoN proteins retain an antiporter function or if they are mere proton channels in Complex I. Some functional specialization of each subunit seems likely, because NuoL more closely resembles MrpA, whereas NuoM and N are more similar to MrpD.8 If these subunits could be overexpressed and purified, the antiporter function could be measured directly in liposomes. All the antiporters and antiporter-like proteins of this protein family are predicted to have the C-terminus in the periplasm8 and can therefore be fused to cytochrome c (Fig. 1). In this work, we have also added
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Table I. Biological Material Bacterial strains E. coli JM109
E. B. B. B.
coli XL1-blue subtilis 168A subtilis DMrpA subtilis DMrpD
Genotype endA1, glnV44, thi-1, relA1, gyrA96, recA1, mcrB D(lacproAB) e14-[F0 traD36 proABþ lacIq lacZDM15] hsdR17(rKmKþ) recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1 (lac) Wild-type (type strain) trpC2 trpC2, DmrpA, Bler trpC2, DmrpD, Bler
Plasmids pUC19 pLUW1900 pCW6 pEC86 pTRC19 pHC1 pLC1 pMC1 pNC1 pVM4 pVM2 pVM11 pVM6 pLch pMch pNch pAch pDch
Amp cccA, Ampr Shuttle vector, Cmr Contains the ccm operon, Cmr Truncated cccA in pUC19, Ampr nuoH fused with truncated cccA, Ampr nuoL fused with truncated cccA, Ampr nuoM fused with truncated cccA, Ampr nuoN fused with truncated cccA, Ampr mrpA fused with truncated cccA, Ampr mrpD fused with truncated cccA, Ampr mrpA fused with truncated cccA, Cmr mrpD fused with truncated cccA, Cmr As pLC1, with his-tag added As pMC1, with his-tag added As pNC1, with his-tag added As pVM4, with his-tag added As pVM2, with his-tag added
35 26 Claes von Wachenfeldt 37 This work This work This work This work This work This work This work This work This work This work This work This work This work This work
5 -GGGCTTGATGACTCTCGGGAG-30 50 -GTATGAGCTCCGCTACTGTTTGC-30 50 -CGCGAAGCTTGAGGATCTCAAGG-30 50 -TTGCGCCTGCCAGAGAATGACAG-30 50 -CCAGAACCTGAAGCTTGATTC-30 50 -ACGCAGTACCATCAACAGTGC-30 50 -CCTGACGAAGCTTTTACAAGGAA-30 50 -CGGCCTTGTAGTAGTAACGGAAT-30 50 -TCCGCGATTGGCAATATCTAGAAGTGG-30 50 -CATCAGCGGCATTGCCAAACGCACA-30 50 -GCAGCTCTTACGTCGACCTATTTTATCGC-30 50 -GCTTTTCCCCTCCTCTTTTACTTTTG-30 50 -CAGGAATTGGAAGCTTACGATATGG 30 50 -CAGAACAGCTTCGATATATTTTTCC-30 50 -CCGCTACTGTCTGCAGTCGTTTA-30 50 -CGCCTTTTCTAGATGCTTTTATC-30 50 -CCGCTACTGTCTGCAGTCGTTTA-30 50 -CGCCAAGCTCTAGATATGGGTC-30 50 P-AAGAACTATTTTTCTCTTTATACATTC-30 50 P-TTAATGATGATGATGATGATGTTTAATTTT TGACACCCACTC-30
Results Construction of the expression vector for cytochrome c fusion proteins The membrane-anchored cytochrome c550 from B. subtilis, described in section ‘‘the choice of cyto-
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Promega Bacillus genetic stock center 36 36
DNA sequence 0
cytochrome c domains to the bona fide antiporters MrpA and MrpD from B. subtilis to use them as antiporter control proteins to compare with NuoL, NuoM, and NuoN.
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Relevant properties r
Primers cccA up cccA down nuoH up nuoH down nuoL up nuoL down nuoM up nuoM down nuoN up nuoN down mrpA_vk_Up mrpA_vk_Dwn mrpD_vk_Up mrpD_vk_Dwn mrpA_For_pCW6 mrpA_Rev_pCW6 mrpD_For_pCW6 mrpD_Rev_pCW6 His-t Forward His-r Reverse
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work work work work work work work work work work work work work work work work work work work
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chrome c,’’ is encoded by the cccA gene. A plasmid for construction and expression of cytochrome-tagged membrane-spanning proteins was produced by cloning the truncated cccA gene, omitting the part of the gene encoding the N-terminal transmembrane helix, into the plasmid pUC19,35 which contains an isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible lac promoter. This plasmid was named pTRC19 (see section ‘‘construction of the pTRC19 plasmid,’’ Table I). In principle, all membrane-spanning proteins with the C-terminal end located in the periplasm can be
Cytochrome c Fusion Proteins for Production of Membrane Proteins
Table II. Cytochrome c550 Content in E. coli Membranes from Different Growth Conditions
Bacteria with plasmid
Conditions (a) 8 h, 37 C, 10 lM IPTG (b) 16 h, 37 C, without induction (c) 17 h, 30 C, 10 lM IPTG (d) 42 h, 30 C, 50 lM IPTG
pHC19 (encoding NuoHcyt) nmol cyt c550/mg membrane protein
pLC19 (encoding NuoLcyt) nmol cyt c550/mg membrane protein
pMC19 (encoding NuoMcyt) nmol cyt c550/mg membrane protein
pNC19 (encoding NuoNcyt) nmol cyt c550/mg membrane protein
pLUW1900 (encoding intact cytochrome c550) nmol cyt c550/mg membrane protein
pUC19 (vector only) nmol cyt c550/mg membrane protein
0.05 0.03 0.04 0.04
nd nd nd 0.55
nd nd nd 0.50
0.14 0.08 0.23 0.46
0.22 0.22 0.08 0.10
