Quantitation of disulfide bonds by fluorescence
An experimental method was developed to visualize the abundance of intracellular protein disulfide bonds in P. aerophilum cell lysates. First, cysteine residues existing in their free thiol form were blocked immediately upon cell lysis by irreversible alkylation with iodoacetamide. Then, any existing protein disulfide bonds were cleaved by chemical reduction. Finally, the resulting free thiol groups were labeled with the thiol-reactive fluorescent reagent CPM. The result of this procedure was a selective labeling of cysteine residues involved in disulfide bonds.
The reliable measurement of disulfide-bonded cysteines in this assay is dependent on the efficiency and specificity of the reagents utilized, and thus careful consideration was given to their selection. Iodoacetamide is well established in redox studies for alkylating free thiols; this blocks cysteine residues in their reduced form and prevents any subsequent oxidation9; 10; 11
. Previous work has demonstrated the high efficiency of this reaction following denaturation of the substrate proteins12; 13
. The fluorescent reagent CPM has also been used extensively in the quantitative analysis of free thiols, and has been shown to exhibit a high degree of specificity to cysteine residues5; 14; 15; 16
. In the current studies, disulfide labeling experiments were analyzed after running SDS gels on the treated cell lysates. As described below, an analysis of the resulting gels supported the veracity of the procedure employed.
The assay revealed an abundance of protein disulfide bonds in P. aerophilum cell lysates (). In order to quantify the abundance of disulfide bonding relative to the total cysteine content in the cellular proteins, the same assay was performed, but with the initial blocking step omitted; this leads to labeling of all cysteine residues. Fluorescence measurements of CPM-labelled proteins in the gels revealed that approximately 47% of the cysteine residues in the P. aerophilum lysate are involved in disulfide bonds, as opposed to approximately 8% in E. coli, which was used as a control. Estimates for the uncertainties in these measured values are 5% and 3%, respectively, based on variations between triplicate experiments; these values account for random but not systematic errors, and so represent lower bounds on the true errors. For E. coli, the measured value for disulfide abundance likely includes contributions from extra-cytosolic (e.g. periplasmic) proteins, which are known to contain disulfide bonds; P. aerophilum cells do not have a periplasmic space.
Figure 1 Abundance of disulfide-bonded proteins in P. aerophilum, as detected by fluorescent labeling. Whole cell lysate was reacted with iodoacetamide (+) to block free (thiol) cysteines. Following blocking, any disulfide bonds present were cleaved by reduction (more ...)
Two kinds of control experiments – the comparison of P. aerophilum to E. coli and the omission of the blocking reagent – were used to address concerns about the efficiency and selectivity of the disulfide labeling protocol. Some of the labeling observed in the gels could reflect non-specific binding of the fluorescent reagent, but the relatively low fluorescence measured for E. coli (8%) suggests that non-specific binding could account for only a few percent of the estimated (47%) disulfide abundance in P. aerophilum. Another concern is that some of the labeling could be to cysteine residues that were not involved in disulfide bonds, but that existed in the free thiol form and were simply not fully blocked in the initial step. It is difficult to eliminate this potential scenario entirely, but two points are noteworthy. First, the cysteine residues in the E. coli proteins were blocked almost completely, as judged by the low overall fluorescent labeling. Second, in the case of P. aerophilum the two lanes in the gel (with and without blocking) show very different patterns. In particular, in the lane in which no blocking was performed, numerous bands are visible that are effectively absent from the lane in which free cysteines were initially blocked by iodoacetamide. The effective disappearance of numerous labeled bands gives a measure of the effectiveness of the blocking step.
These new experimental results on whole cell lysates support earlier claims that disulfide bonding is widespread in P. aerophilum
proteins5; 6; 17
. The results also suggested the value of further experiments aimed at analyzing individual P. aerophilum
proteins and the nature of their disulfide bonds.
Identification of disulfide-bonded protein complexes by 2D diagonal gel electrophoresis
An experimental method employing 2-dimensional diagonal gel electrophoresis (2D-DGE) was developed for identifying protein complexes in P. aerophilum
that might be held together by disulfide bonding between protein chains (). Similar methods have been described for identifying ribosomal proteins with non-native intermolecular disulfide bonds18
, identifying protein targets of DsbA11
, investigating oxidative folding in the endoplasmic reticulum19; 20
, and most recently for identifying cytosolic proteins that form stable disulfide bonds under oxidative stress21; 22
. In 2D-DGE, proteins are initially separated (in the horizontal dimension) by SDS-PAGE under non-reducing conditions that preserve any existing disulfide bonds. Prior to separation in the second (vertical) dimension, proteins within the gel are chemically reduced, cleaving any disulfide bonds that are present. Proteins that do not contain disulfide bonds migrate at the same rate in both dimensions, creating a prominent diagonal line in the 2D gel. Spots below the diagonal arise from proteins that were held together in disulfide-bonded complexes within the cell. Such proteins migrate more rapidly in the second dimension because the complexes in which they were held during the first electrophoresis have been separated into their smaller components. In addition, some individual proteins bearing intramolecular disulfide bonds appear above the diagonal. Such proteins can have mobilities that are measurably higher in the first dimension owing to a lower average extension of the protein chain when constrained by an intramolecular disulfide bond. As shown in -DGE analysis of a P. aerophilum
lysate revealed numerous off-diagonal spots corresponding to disulfide bonded proteins. In particular, a large number of protein-protein complexes apparently held together by intermolecular disulfide bonds were observed below the diagonal. As expected, control experiments with E. coli
revealed very few proteins off the diagonal in the 2D gel ().
Figure 2 A 2-D diagonal gel electrophoresis method for identifying intermolecular disulfide bonded protein complexes. The first separation (1) is performed under non-reducing conditions so that disulfide bonds remain intact. Disulfide bonds are cleaved by reduction (more ...)
To identify which proteins participate in disulfide bonding, off-diagonal protein spots were excised, subjected to in-gel trypsin digestion, and analyzed by liquid chromatography-tandem mass spectrometry. Identifications based on genomic sequence data were successful for 16 spots (), with the predicted molecular weights of identified proteins consistent with the observed molecular weight of the associated spot. All identified proteins contained at least one cysteine residue, while proteins predicted to form intramolecular disulfide bonds contained at least two cysteines. Ten of the proteins were annotated in the NCBI database as hypothetical or conserved hypothetical, making it difficult to assign cellular functions to them. However, an analysis of homologous sequences23
from other organisms showed that the majority of the cysteines in the identified proteins are not conserved. This suggests that the cysteines residues are not involved in enzymatic functions, but rather serve structural roles as anticipated. The major spot observed in the E. coli
gel (EC1) was identified as an alkyl hydroperoxide reductase (AhpC), a homodimeric enzyme known to form an intermolecular disulfide bond during reduction of organic hydroperoxide24
Disulfide bonded P. aerophilum proteins and complexes identified from 2D gels.
A number of the weaker spots in the P. aerophilum 2D gels could not be identified by mass spectrometry. Furthermore, there are likely additional disulfide bonded protein complexes in P. aerophilum that are present at concentrations too low to be visualized in the 2D gels. The proteins identified () therefore represent only a subset of those that form disulfide bonded complexes in P. aerophilum. Conversely, based on the amino acid sequences and molecular mass data from the gels, none of the proteins identified as being in disulfide bonded complexes appear to be false positives. The method employed here may be useful in the future for identifying disulfide bonded complexes in other hyperthermophilic organisms. However, the feasibility of broad-based studies is limited at the present time by the general difficulty associated with culturing such organisms.
One of the protein spots observed in the P. aerophilum
2D-DGE analysis was identified as citrate synthase. Citrate synthase has been developed as a model protein for structural studies of thermophilic adaptation25
, with crystal structures described for psychrophilic, mesophilic, thermophilic, and hyperthermophilic homologues26; 27; 28; 29; 30
. Among the numerous homologues studied, none contain disulfide bonds, making the P. aerophilum
citrate synthase (PaCS) the first shown to utilize this mechanism. In an effort to further characterize the enzyme and analyze the stabilizing effects of its disulfide bonds, PaCS was cloned, expressed, and purified for biochemical and structural studies. The recombinant protein was found to form a homodimeric complex, as anticipated from the 2D gel analysis and in agreement with the known structures of homologous enzymes from other organisms. In addition, SDS-PAGE analysis in the presence and absence of reducing reagent confirmed that the dimeric state was dependent on the presence of disulfide bonds (), consistent with the 2D-DGE observations.
Figure 5 Intramolecular disulfide bonds leading to topological linkage or catenation of protein chains in P.aerophilum citrate synthase (PaCS). (A) Cartoon representation of interlinked PaCS chains. On the left, the protein backbone of the PaCS dimer is shown (more ...)
Crystal structure of citrate synthase
The protein was crystallized and its structure was determined to a resolution of 1.6Å. The PaCS structure is dimeric, with each protein subunit consisting of 18 α-helices and 8 β-strands (). The structure overlaps well with previously determined citrate synthase structures, consistent with the strong conservation of the protein from bacteria to mammals. Each subunit is composed of one large domain (helices C-M and S), one small domain (helices N-R), and additional structural features at the termini (). The active site of citrate synthase, identified through conserved active site residues, resides between the large and small domains of each subunit. The C-terminal region of each PaCS subunit wraps across the opposite subunit in a manner reminiscent of domain swapping31
, with residues from the C-terminal arm contributing to the active site of the opposing subunit. Thus, stability of the dimer is necessary for enzyme activity.
Figure 3 Crystal structure of P. aerophilum citrate synthase (PaCS). (A) The PaCS homodimer illustrated with the individual subunits colored red and blue. The arrangement of domains is illustrated for the (B) PaCS dimer, (C) individual subunit, and (D) PaCS dimer (more ...)
The N-terminal extension
Despite the general similarity of PaCS to homologues from other species, structural and sequence comparisons reveal unique features in PaCS (). In particular, PaCS contains an N-terminal extension not present in any of the archaeal or bacterial structures currently known. The pig and chicken citrate synthases also contain an N-terminal extension, but the sequence and structural features do not resemble those of PaCS. The PaCS N-terminal extension encodes three β-strands (strands 1–3). An intermolecular antiparallel β-sheet is formed between strand 1 and strand 1’ from the other subunit, while strands 2 and 3 interact intramolecularly to form another small antiparallel β-sheet within each subunit. These interactions suggest that the N-terminal extension serves to stabilize the dimer against thermal denaturation. In addition, the loop between strands 2 and 3 contains one of the cysteine residues (Cys19) involved in the key disulfide bond.
Figure 4 A multiple sequence alignment of citrate synthase homologues from four thermophilic organisms, illustrating the unusual N-terminus in P. aerophilum. Overall conservation in the remainder of the protein is highlighted with α-helices shaded in grey (more ...)
Disulfide bond formation leads to catenation
Based on the biochemical results, it was anticipated that the disulfides present in PaCS should form intermolecular disulfide bonds, as the oxidized form of the protein ran as a dimer during eletrophoresis under denaturing conditions. An analysis of the structure confirmed the presence of disulfide bonds, but revealed that the disulfides are in fact intramolecular, between Cys19 and Cys394 from the same subunit (). The formation of this internal bond within each subunit confers a topological connection between the subunits. Though not directly bonded, the two subunits are connected like links in a chain, making the dimer an example of what has been called a protein catenane. This interlinking of cyclized protein chains renders them inseparable even under denaturing conditions ().
Interestingly, the N-terminal extension and disulfide-forming cysteines are conserved in the closely related Thermoproteus tenax and Pyrobaculum islandicum citrate synthases. These homologues have not been characterized biochemically or structurally, but the sequence conservation suggests that the topological linkage observed in P. aerophilum may be a feature common to this branch of archaeal homologues. Additional sequence and structure data should help clarify the evolutionary history of the unusual features of this enzyme.
Disulfide bonded catenation contributes to thermal stability
The stabilizing effect of the disulfide bonds was analyzed by comparing the native protein (PaCSnat) to a mutant version (PaCSmut) in which the two cysteines were replaced by serine residues. Due to the high stability of both proteins, addition of the denaturant guanidinium-HCl (GdnHCl) was required to partially destabilize the proteins in order to observe their unfolding transitions by circular dichroism. Although both constructs exhibited similar CD profiles at room temperature, PaCSnat exhibited a melting temperature (Tm) of 84°C, while PaCSmut exhibited a Tm of 73.5°C, a decrease of 10.5°C from the PaCSnat (). This shift in melting temperature suggests that the linkage created by the Cys19-Cys394 disulfide bond plays an important role in stabilizing the protein against thermal denaturation. Interestingly, enzymatic activity assays showed no difference between the reaction rates of PaCSnat and PaCSmut at temperatures up to 90°C (data not shown), indicating that other stabilizing factors, in addition to the disulfide bonds, contribute greatly to the high stability of the dimeric complex.
Figure 6 Thermal denaturation of native and mutant forms of P. aerophilum citrate synthase in 4.5M Gdn-HCl, monitored by circular dichroism. Experiments in the oxidized (thin curve) and reduced forms (thick curve) illustrate the stabilizing contribution from the (more ...)
Comparison of PaCS to homologous structures
Analysis of the native and mutant proteins revealed the stabilizing effect of the disulfide bond. However, the high stability of the disulfide-free mutant indicates that other factors are also important to the stability of the enzyme. Previous comparative studies of citrate synthase structures from distantly related organisms have identified several subtle trends correlated with thermophilicity, including decreased surface area and volume, shortening of loops, and increased ion pairing 25; 32
. An analysis of these trends with respect to the PaCS structure revealed key differences in how certain stabilizing mechanisms are utilized between the various homologues. summarizes several properties associated with thermal adaptation for a set of citrate synthase homologues. Previous studies highlighted an overall decrease in surface area and volume for thermophilic citrate synthases relative to the mesophilic pig citrate synthase (PigCS). Surprisingly, this trend does not hold for PaCS, as both the surface area and volume were found to be greater for PaCS than for all but the PigCS structure. The increase in size is partly due to the N-terminal extension and additional α-helices that are unique to the PaCS structure. The contributions of these elements to stability may override the potentially destabilizing effect of increased size. PaCS also exhibited a shortening of loops between α-helices. This decrease in disordered regions is likely to help counter the increase in size33
. To take into account these differences in chain length, we analyzed the surface area and volume per residue. Interestingly, the PaCS still showed a greater surface area and volume per residue but, more significantly, the trend of decreased surface area and volume as a function of thermophilicity was no longer apparent, with the volume per residue actually greater in thermophilic homologues. These results show that decreased surface area and volume are not strongly correlated with increased thermal stability.
Comparison of structural features in homologues of citrate synthase.
Intermolecular ion-pairing and hydrophobicity at the dimeric interface have previously been implicated as stabilizing elements in the thermophilic citrate synthase homologues. An analysis of overall hydrophobic and charged amino acid content showed no correlation with thermophilicity, emphasizing the importance of three-dimensional spatial considerations in understanding thermal stability. Interestingly, a structural comparison of PaCS and PfCS revealed significant differences in how various stabilizing elements are utilized. Although ion-pairing was previously identified as a major stabilizing mechanism at the PfCS dimeric interface 25
, it does not appear to play as great a role in the stabilization of the PaCS dimer; only 8% of the residues in the P. aerophilum
interface are charged, while the corresponding value is 22% for P. furiosis
. Ion pairs in PaCS seem to be primarily intramolecular, stabilizing each subunit. Instead, PaCS utilizes hydrophobic interactions as a primary mechanism for stabilizing the dimeric interface. In P. aerophilum
, 62% of the residues in the dimeric interface are hydrophobic (following the definition in ), while the corresponding value is only 32% for P. furiosis