(Received for publication, July 17, 1995; and in revised form, August 23, 1995)
From the
The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3`-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 °C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
Bovine heart mitochondrial ubiquinol-cytochrome c reductase, also known as complex III, or the cytochrome b-c complex, catalyzes electron transfer from
ubiquinol to cytochrome c
with concomitant
transfer of protons across the membrane to generate a proton gradient
and membrane potential for ATP synthesis(1) . This complex has
recently been crystallized (2, 3, 4) and
shown to contain ten protein subunits (2) with five redox
centers. The molecular mass of these subunits (I to X numbered as
decreasing molecular mass) are 51, 49, 43, 27.9, 21.5, 13.4, 9.5, 9.2,
8, and 7.2 kDa. The redox centers are as follows: two b cytochromes (b
and b
), one c-type cytochrome (c
), one high potential iron-sulfur cluster
(Rieske 2Fe-2S cluster), and one ubiquinone.
The amino acid
sequences of all the protein subunits are available from gene (5, 6, 7) or protein
sequencing(8, 9, 10) . Subunits I and II are
identified as core proteins. Subunits III-V are cytochrome b, cytochrome c, and iron-sulfur protein,
respectively. Controlled digestion studies with trypsin suggest that
subunit VI is involved in redox-linked proton pumping(11) .
Subunit VII (QPc-9.5 kDa), together with subunit III (cytochrome b), are identified as Q
-binding proteins by
photoaffinity labeling using azido-Q derivatives(12) . Subunit
VIII (also known as hinge protein) is tightly associated with
cytochrome c
and may facilitate the binding of
cytochrome c to cytochrome c
(13) . Subunit IX has been shown to bind DCCD and may be
involved in proton translocation(14) . The functional role of
subunit X is unknown.
The electron transfer mechanism of
ubiquinol-cytochrome c reductase is consistent with the
Q-cycle scheme originally proposed by Mitchell (15) and
subsequently refined by Berry and Trumpower(16) . In the
Q-cycle, ubiquinol is oxidized at center o. One electron from ubiquinol
is transferred to iron-sulfur protein, which then reduces cytochrome c. The second electron is transferred from
ubisemiquinone to cytochrome b
. The reduced
cytochrome b
gives an electron to cytochrome b
, which then reduces ubiquinone at center i.
The identification of two Q-binding proteins in ubiquinol-cytochrome c reductase is consistent with the two Q-binding sites
(Q
and Q
) proposed in the Q-cycle. Whether the
two Q-binding proteins form two binding sites corresponding to Q
and Q
, individually or in combination, remains to be
elucidated.
To better understand the Q-mediated electron transfer
mechanism in mitochondrial ubiquinol-cytochrome c reductase
requires knowledge of the molecular structure of the Q-binding site(s).
The Q-binding domains in cytochrome b and QPc-9.5 kDa have
recently been identified by isolating and sequencing Q-peptides from
[H]azido-Q-labeled proteins. They are located at
amino acid residues 142-155 and 326-336 of cytochrome b(17) and residues 48-57 of QPc-9.5
kDa(18) .
More detailed knowledge of the amino acid residues involved in Q-binding is needed if we are to understand the molecular structure of the Q-binding site. Herein we report the cloning and nucleotide sequencing of a cDNA encoding QPc-9.5 kDa, construction of a QPc expression vector, pGEX/QPc, and development of optimal conditions for high expression of an active soluble form of the GST-QPc fusion protein in Escherichia coli JM109. Isolation and characterization of pure recombinant QPc-9.5 kDa are also reported.
E. coli strains were grown in LB
medium(19) . When necessary, MgSO (10 mM),
maltose (0.2%), tetracycline (15 µg/ml), and ampicillin (50
µg/ml) were added.
Protein concentration was determined by the Lowry et al.(23) method using bovine serum albumin as a standard. Analytical SDS-PAGE was performed in a Bio-Rad Mini-Protean dual slab cell using the gel system of Laemmli (24) or of Schägger et al. (25) with modifications(18) . Western blotting was performed as described previously(18) .
Figure 1: Nucleotide and deduced amino acid sequences of QPc-9.5 kDa. The N-terminal glycine residue of the mature protein is numbered 1. Tryptophan 61 is the only tryptophan residue in the QPc-9.5 kDa. This residue was identified as a cysteine by protein sequencing(10) .
Figure 2:
Proposed structure of QPc-9.5 kDa. The
model was constructed from the hydropathy profile of the deduced amino
acid sequence, and the predicted tendencies to form -helices and
-sheets.
Figure 3: Construction of the QPc-9.5 kDa expression vector, pGEX-QPc.
Recovery of active recombinant protein from the inclusion body complexes has long been regarded as a formidable task due to the denaturation of the protein and the heterogeneous nature of inclusion bodies(29) . Although general techniques for obtaining active recombinant protein from inclusion bodies have not been developed, some success has been reported(30, 31, 32) . In these cases, recombinant protein aggregates were solubilized with a high concentration of urea, or other chaotropic reagents, and followed by dialysis, to allow proper refolding of the protein structure(32) . When insoluble aggregates of recombinant GST-QPc were treated with 8 M urea, about 75% of the fusion protein was solubilized. However, when the urea was removed by dialysis, more than 95% of the protein precipitated. When the remaining soluble protein was applied to a glutathione agarose gel, no protein was bound to the gel, indicating that the GST-active structure had not properly re-folded. The failure to regenerate active soluble recombinant GST-QPc from inclusion bodies by the urea-dialysis method suggests that this approach is difficult or impossible for GST-QPc fusion protein. Therefore, development of an environment conducive to production of active, soluble GST-QPc recombinant protein by E. coli was necessary if the pGEX/glutathione agarose system was to work for this investigation.
Changes in cellular environments are known to increase the yield of active recombinant protein either by encouraging the cells to adopt the active conformation or by increasing the stability of recombinant proteins. Variation of media, induction conditions, and length of induction are factors that affect the cellular environments of E. coli. We systematically examined the effect of these factors on the yield of soluble recombinant GST-QPc fusion protein. Fig. 4compares the yield of recombinant GST-QPc fusion proteins eluted from glutathione agarose gel, after incubation with soluble fractions of lysate from E. coli culture induced at 37 °C (lane 3) and 27 °C (lane 4). Induction at 27 °C improved the yield over induction at 37 °C, by 4-fold, although the total amount of recombinant GST-QPc produced by the E. coli cultures induced at these temperatures was about the same. In other words, when the cell culture was induced at 27 °C, about 40% of the GST-QPc fusion protein produced was in the soluble form, compared to 10% with the culture induced at 37 °C. This result is compatible with previous reports (32, 33) of increased soluble yield of recombinant polypeptide in E. coli using low growth temperature.
Figure 4: Recovery of purified GST/QPc fusion protein from cells grown at various conditions. A, SDS-PAGE of glutathione agarose gel eluates obtained from cells after the IPTG induction growth at 37 °C (lane 3) and 27 °C (lane 4) in LB medium containing 50 µg/ml ampicillin. Lane 5 is the same as lane 4, except 2.5 mM betaine and 440 mM sorbitol were added to the medium. Lane 2 represents cells growth without IPTG. Lane 1 is molecular weight references. The isolation of GST/QPc fusion protein by affinity glutathione agarose gel was as described under ``Experimental Procedures.'' 30 µl of glutathione agarose eluates were applied to SDS-PAGE. B, the proteins on the gel of A were electrophoretically transferred to a nitrocellulose membrane without staining and then reacted with anti-QPc antibodies. Alkaline phosphatase conjugate was used as a second antibody.
Although lowering the induction temperature increased the yield of soluble active GST-QPc fusion protein, about 60% of the recombinant fusion protein still remained in inclusion body complexes. To further increase the soluble yield, a method involving the use of osmotic stress to facilitate the uptake of the ``compatible solute'' glycyl betaine (34) was adopted. When E. coli JM109/pGEX-QPc cells were grown at 27 °C on LB medium containing 2.5 mM betaine and 440 mM sorbitol, the yield of soluble recombinant protein was twice as high as that for cells grown in the absence of these compounds (see Fig. 4, lanes 4 and 5). This result is similar to that reported by Blackwell and Horgan (35) in which the inclusion of betaine and sorbitol in the growth medium converts a highly expressed Agrobacterium DMAPP:AMP transferase, which normally accumulates as inclusion complexes in E. coli, into an active soluble form. Although the reason for the increased yield of active soluble recombinant protein is unknown, it has been suggested (36) that increasing internal concentrations of compatible osmolytes, such as betaine, which are believed to be excluded from the immediate domains of proteins, causes a thermodynamically unfavorable ``preferential hydration'' and thus minimization of solvent-protein contact and stabilization of protein structure.
Production of active soluble recombinant GST/QPc fusion protein was found to be IPTG-induction growth time dependent (data not shown). The yield increased as the induction growth time was increased, to a maximum yield which was obtained when cells were harvested three hours after growth induction. When cells were grown for 5 h, there was a 20% decrease in yield, suggesting that the recombinant protein is unstable and susceptible to protease digestion. In summary, the optimal conditions for the production of soluble active recombinant GST/QPc fusion protein are as follows: induction of E. coli JM109/pGEX-QPc cells with IPTG at 27 °C for three hours on LB medium containing 2.5 mM betaine and 440 mM sorbitol.
Our routine cell extract procedure included treatment with 1% Triton X-100. If this treatment was omitted, the yield of soluble recombinant GST-QPc fusion protein decreased 30%. This suggests that some GST-QPc fusion protein is either in a membrane fraction or an inclusion body aggregate that can be solubilized by Triton X-100 while maintaining the GST-active site recognizable by glutathione agarose gel. Recombinant GST-QPc fusion protein obtained from cell extracts prepared with Triton X-100 has the same molecular size and Q-binding properties as that obtained without Triton X-100 treatment.
Although isolated recombinant QPc-9.5 kDa is soluble in
aqueous solution, it has a molecular mass of over 1 million as
determined by FPLC gel filtration with Sepharose-12 in 50 mM
Tris-Cl buffer, pH 8.0. Aggregation is apparently due to the
hydrophobic transmembrane segment of the peptide chain. The hydropathy
plot of QPc suggests a definitive transmembrane helix. In the presence
of dodecyl maltoside and 300 mM NaCl, aggregated recombinant
QPc is dissociated into the monomeric form. The soluble hydrophobic
aggregation of isolated QPc resembles isolated mitochondrial cytochrome c, which exists as pentamers and is soluble in
aqueous solution(37) . Isolated QPc gives a single protein band
in SDS-PAGE which corresponds to subunit VII (9.5 kDa) of mitochondrial
ubiquinol-cytochrome c reductase (see Fig. 5).
Actually, the partial N-terminal amino acid sequence of recombinant QPc
was determined to be GSMGRQ-, indicating that three additional amino
acid residues, glycine, serine, and methionine are present at the N
terminus of mature QPc-9.5 kDa. The glycine and serine residues result
from the recombinant manipulation whereas methionine, which is encoded
by the starting codon, ATG, of QPc-9.5 kDa, is post-translationally
cleaved from the native mature protein. The true molecular mass of
recombinant QPc should be 275 daltons more than that of mature
mitochondrial QPc-9.5 kDa.
Figure 5: SDS-PAGE of isolated recombinant QPc-9.5 kDa. Lane 1, molecular weight standard; lane 2, bovine heart mitochondrial ubiquinol-cytochrome c reductase; lane 3, purified recombinant QPc. The high resolution SDS-PAGE gel of Schägger et al.(25) was used.
With pure recombinant protein available,
the simplest approach for investigating its functional activity would
be to reconstitute it with QPc-depleted ubiquinol-cytochrome c reductase and determine whether or not the enzymatic activity or K for Q
H
of the complex is
restored. Unfortunately, reconstitutively active, QPc-depleted (or
deficient) ubiquinol-cytochrome c reductase is not yet
available. Therefore we took an alternative approach, measurement of
the ability of the recombinant protein to bind Q. This approach is
based on the assumption that binding of Q to its binding protein will
affect the spectral properties of Q, and if the binding is specific, a
titration curve should exhibit a saturation behavior.
When Q was added to a recombinant QPc-9.5 kDa solution containing 10% ethanol, a blue spectral shift was observed (see Fig. 6). The spectral blue shift of Q was not detected when a Pronase-treated recombinant QPc-9.5 kDa was added. Titration of recombinant QPc-9.5 kDa with Q showed a saturation point at around 2 moles of Q per mole protein. These results suggest that the binding of Q to recombinant protein is specific and the recombinant QPc-9.5 kDa may be functionally active. To confirm the specific binding between recombinant QPc-9.5 kDa and Q, two other proteins, bovine serum albumin and ribonuclease A, were used as controls in the titration studies. Ribonuclease A had absolutely no effect on absorption spectral properties of Q, suggesting no interaction between them. Since bovine serum albumin is known to bind hydrophobic compounds such as fatty acid, a nonspecific binding of this protein with Q is expected. As shown in Fig. 6, the effect of bovine serum albumin on spectral properties of Q is very much different from that observed with recombinant QPc-9.5 kDa.
Figure 6:
Effect
of recombinant QPc-9.5 kDa on absorption maximum of Q. 0.8-ml aliquots
of 20 mM TrisCl, pH 8.0, containing 10% ethanol, 150 mM NaCl, and 2.5 mM CaCl were added to 50 µl
of 50 mM Tris-Cl buffer, pH 8.0, containing none (
),
ribonuclease A (
), bovine serum albumin (
), recombinant
QPc-9.5 kDa (
), or Pronase-treated recombinant QPc-9.5 kDa
(
). The final protein concentration was 130 µg/ml. Each
sample as prepared in duplicate and placed each in a 1-ml cuvette (1-cm
light path). Q
C
Br in 95% ethanol was added to
one sample cuvette in 0.5-µl increments to obtain the
concentrations indicated. At the same time, alcohol (95%) was added to
the second sample cuvette in a identical manner. For each addition,
after incubation for 5 min, a difference spectra between
Q
C
Br-added and alcohol-added samples were
recorded from 320 to 250 nm.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L06665[GenBank].