The successful purification of proteins of interest to homogeneity is the first critical step in their detailed characterization. The purification of mutant forms of proteins has its own challenges due to the effect of the mutation on their native properties. Recently, we have reported a successful purification and characterization of wild type and eight mutant CBS enzymes [16
]. The CBS enzymes were expressed as fusion proteins with a GST partner at the N-terminus, which after cleavage with the HRV3C (also known as PreScission) protease and subsequent chromatographic separation of the GST yielded a ≥90% pure CBS polypeptide with only a single extra amino acid residue, glycine, at the N-terminus of CBS compared to the native sequence. We have used the same expression/purification system to prepare CBS enzyme containing cobalt protoporphyrin instead of heme in order to unravel the role of the heme cofactor in CBS enzymes from higher eukaryotes [23
]. However, several mutant forms of CBS including R266K eluded our efforts of purification and characterization using this procedure. Our data presented here shows the GST-tagged R266K CBS mutant forms inactive aggregates, which precluded its purification. To our surprise, Singh et al. [20
] reported a successful purification and spectroscopic characterization of an R266K CBS mutant using a GST-based system. However, compared to our GST-CBS construct the spacer between the GST fusion partner and CBS was substantially longer and after cleavage with thrombin protease, the final CBS carried an extra 11 amino acid residues. These opposing results, using similar expression systems, suggested that although our construct yields a CBS polypeptide as similar to the native CBS sequence as possible, which works for wild type as well as many CBS mutants, the presence of GST likely interferes with the folding and/or proper assembly of the CBS polypeptide for some mutants, such as R266K. The extra 11 residues in the construct of Singh et al. [20
] most likely prevented undesired interactions between GST and CBS. The potential disadvantage of an extended spacer between GST and CBS is an alteration of some of the enzymes properties. Janosik et al. [3
] reported a significantly lowered (~9-fold) Km
of CBS, with 23 extra residues at the N-terminus, for homocysteine. Altogether, the data suggests that there was a need for a new construct, which would allow for efficient expression and purification of both wild type and mutant CBS. Such construct(s) should avoid the use of a bulky fusion partner such as GST, which may interfere with the structural properties of CBS polypeptides, and at the same time should yield the CBS sequence as close to its native sequence as possible to avoid alterations in the biochemical properties of the purified CBS enzymes.
We prepared three CBS constructs carrying a short affinity (6xHis) tag and evaluated their efficacy for the production of pure wild-type and mutant CBS enzymes. Generally, the small size of the 6xHis tag does not interfere with the folding, assembly or biochemical properties of the partner protein [26
]. Recently, a new cost-effective and efficient expression/purification system for CBS based on a short affinity (6xHis) tag has been reported [28
]. The authors reported that the CBS construct bearing the C-terminal 6xHis tag was insoluble probably due to a destabilization of the CBS oligomeric structure, which resulted in aggregation and precipitation. In our hands, the C-terminally 6xHis-tagged CBS construct yielded a soluble CBS polypeptide. Moreover and in stark contrast to the previously reported study [28
], our construct yielded more active tetrameric CBS enzyme in crude extract than any other 6xHis-tagged or GST-based constructs tested herein. The plausible explanation of such different results may possibly involve a failure to verify the DNA sequence or a problem with expression and/or purification of Belew’s et al. construct [28
The presence of chemical chaperones such as ethanol, TMAO or DMSO during CBS expression was recently shown to facilitate proper folding and to rescue the activity of several CBS mutants [15
]. We have also shown that the effect of chemical chaperones may be indirect by increasing the steady-state levels of molecular chaperones, such as DnaJ, a microbial analog of HSP40 [16
]. The lowering of the temperature from 37°C to 18°C during CBS expression had a similar effect as the presence of chemical chaperones on recovery of native tetramers and activity of several CBS mutants [21
]. This data suggests that the deficiency in CBS activity may be in some instances caused by misfolding that may be alleviated by folding under permissive conditions. The R266K CBS mutant was found to be responsive to a chaperone treatment by inclusion of betaine or glycerol in the growth medium or to the lowering of the temperature during bacterial growth and CBS expression [17
]. In this study we demonstrated that the presence of ethanol, TMAO or DMSO as chemical chaperones did not promote a significant increase in R266K CBS activity when expressed from a GST-based or 6xHis-based construct. However, we detected an increased amount of native tetramer for the C-terminally 6xHis-tagged R266K CBS, which indicates that chemical chaperones promoted proper folding and/or tetramer assembly of this protein. The lack of correlation between the increased amount of native tetramer and no significant changes in the specific activity of R266K may be explained by a topology of this particular mutation. According to the crystal structure of the truncated form of human CBS, the R266 is a residue buried in the structure of the CBS globule [5
]. Kozich et al. [21
] showed that several buried mutations, particularly G307S, lacked the correlation between the amount of native tetramers and specific activity of the CBS mutant. On the other hand, increased activity of several CBS mutants containing a solvent-exposed mutation mirrored the increased amount of tetramers suggesting that solvent-exposed mutations are more likely to respond to interventions aimed at correcting their impact on CBS polypeptide folding.
The R266 is not just a buried residue within the CBS structure, but also an important residue in the second coordination sphere of the axial heme ligand C52. Singh et al. [20
] showed that the electrostatic interaction between R266 and C52 is critical for the stabilization of the ferrous heme and its disruption leads to the irreversible formation of an inactive ligand-switched species. The relevance of their finding is somewhat questionable since it is not known whether CBS heme undergoes redox changes in vivo
and thus whether there is any heme-based allosteric redox regulation of CBS activity. Here we showed that the R266K mutation affects (i) the enzyme saturation with a PLP cofactor, (ii) the response to the CBS allosteric activator AdoMet and (iii) the thermal stability compared to wild type. The modest increase in CBS mutant activity after addition of exogenous PLP correlates well with the results reported by Singh et al. [20
]. The extent of CBS response to AdoMet varies greatly and partially correlates with the folding, assembly of tetramers and particularly with the relative position of the catalytic core to its C-terminal regulatory region. In addition, the thermal pre-treatment of CBS prior to an activity assay mimics the enzyme activation with AdoMet [3
]. Our data showed that the response of the R266K CBS mutant to AdoMet is significantly lower compared to wild type. Moreover, thermal pre-treatment resulted in only a modest increase in activity and then began to decrease in activity after 55°C possibly due to premature aggregation.
Indeed, the R266K mutant formed aggregates at a lower temperature than wild type suggesting its decreased thermal stability. The differences at 430 nm, monitoring the Soret peak of the CBS heme, between the wild type and mutant spectra may be plausibly explained by a premature aggregation of the mutant enzyme. The decrease of Soret peak intensity for wild type enzyme with increasing temperature is most likely due to the loss of the heme ligand, which converts the heme from a low-spin into a high-spin species [31
]. The subsequent increase in 430 nm absorbance with increasing temperature is most likely due to protein aggregation, which increases the baseline absorbance. In R266K, the loss of the heme ligand was either not present or masked by a more facile aggregation. We assume that the R266K heme environment, where the pathogenic mutation already affects the stability of the heme thiolate ligand, is more sensitive to heme ligand loss with increasing temperature than wild type, which in turn immediately triggers protein aggregation.
Our previous studies showed that heme is essential for the expression of an active human CBS and that the saturation of enzyme with heme cofactor directly correlates with an amount of bound PLP and thus with CBS catalytic activity [33
]. Destabilization of CBS heme’s second coordination sphere by the R266K missense mutation may be communicated to the PLP-containing active site of CBS via α-helix 8 and possibly resulting in premature irreversible loss of PLP cofactor in the partially unfolded enzyme by the thermal pre-treatment. Such a link would further confirm the importance of an intact connection between heme and PLP in human CBS. Detailed spectroscopic characterization of the R266K heme environment will be a subject of another study.