Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.
Immobilisation of enzymes onto surfaces with defined orientation is of particular importance in order to optimise their activity and capacity to dock electron‐transfer partners. Local characterisation methods are thus crucial to understanding the activity–orientation relationship of immobilised enzymes for long‐term storage and applications. Herein, cytochrome c (cyt c) is chemoselectively immobilised through a cysteine functional group with a maleimide‐functionalised gold surface, thus controlling cyt c orientation. X‐ray photoelectron spectroscopy confirmed successful cyt c immobilisation. Scanning electrochemical microscopy methodologies were developed to quantify the activity of immobilised cyt c and cytochrome c peroxidase (CcP). The results suggest optimal enzymatic activities and docking ability for surface‐tethered cyt c are obtained through the cysteine–maleimide linkage, in comparison to conventional indiscriminate lysine–carboxyl linkages. This study reveals the strong influence of cyt c orientation, as a result of linking strategy, upon the docking ability of redox‐electron partner CcP.
Rationale The decolouration of brilliant blue FCF by the action of titanium dioxide (TiO2) under ultraviolet (UV) exposure has been recently reported as the basis of a paper‐based sensor for monitoring UV sun exposure. The mechanism of brilliant blue FCF photodegradation in the presence of the photocatalyst and the resulting photoproducts are thus far unknown. Methods The UV‐initiated photodegradation of brilliant blue FCF in the presence of TiO2 for both the aqueous and the solid state was investigated. Degradation in the solid state was observed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐MS). Decomposition of the dye in the aqueous state was analyzed using liquid chromatography/mass spectrometry (LC/MS) and ultraviolet–visible (UV–Vis) spectroscopy. Results After UV radiation exposure, the brilliant blue FCF base peak [M1 + NH4]+ (m/z calc. 766.194 found 766.194) decreased in the LC/MS chromatogram with a concomitant appearance of BB‐FCF decomposition products involving the sequential loss of the N‐ethyl and N‐methylbenzene sulfonate (MBSA) groups, assigned as [M2 + H]+ (‐MBSA, calc. 579.163 found 579.162), [M3 + H]+ (‐MBSA, −Et, calc. 551.131 found 551.131), [M4 + H]+ (‐2MBSA, calc. 409.158 found 409.158), [M5 + H]+ (‐2MBSA, −Et, calc. 381.127 found 381.127). Ions [M2 + H]+ and [M3 + H]+ were also identified in the photodegradation products using MALDI‐MS. Observation by UV–Vis indicated a decrease in the solution absorbance maxima and an associated blue‐shift upon UV exposure in solution. Conclusions The LC/MS analysis indicated two main oxidation processes. The most obvious was attack of the N‐methylene, eliminating either ethyl or MBSA groups. The presence of the hydroxylated decomposition product M13 ([M13 + H]+, calc. 595.157 found 595.157) supported this assignment. In addition, the detection of photoproduct M8, proposed to be 3‐((ethylamino)methyl)benzenesulfonic acid ([M8 + H]+, calc. 216.069 found 216.069), indicates an aryl‐oxidative elimination. The absence of the aryl‐hydroxy products normally expected to accompany the formation of M8 is proposed to be due to TiO2‐binding catechol‐like derivatives, which are then removed upon filtration.
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