Semi-enzymatic acceleration of oxidative protein folding by N-methylated heteroaromatic thiols

We report the first example of a synthetic thiol-based compound that promotes oxidative protein folding upon 1-equivalent loading to the disulfide bonds in the client protein to afford the native form in over 70% yield. N-Methylation is a central post-translational processing of proteins in vivo for regulating functions including chaperone activities. Despite the universally observed biochemical reactions in nature, N-methylation has hardly been utilized in the design, functionalization, and switching of synthetic bioregulatory agents, particularly folding promotors. As a biomimetic approach, we developed pyridinylmethanethiols to investigate the effects of N-methylation on the promotion of oxidative protein folding. For a comprehensive study on the geometrical effects, constitutional isomers of pyridinylmethanethiols with ortho-, meta-, and para-substitutions have been synthesized. Among the constitutional isomers, para-substituted pyridinylmethanethiol showed the fastest disulfide-bond formation of the client proteins to afford the native forms most efficiently. N-Methylation drastically increased the acidity and enhanced the oxidizability of the thiol groups in the pyridinylmethanethiols to enhance the folding promotion efficiencies. Among the isomers, para-substituted N-methylated pyridinylmethanethiol accelerated the oxidative protein folding reactions with the highest efficiency, allowing for protein folding promotion by 1-equivalent loading as a semi-enzymatic activity. This study will offer a novel bioinspired molecular design of synthetic biofunctional agents that are semi-enzymatically effective for the promotion of oxidative protein folding including biopharmaceuticals such as insulin in vitro by minimum loading.

Semi-enzymatic acceleration of oxidative protein folding by N-methylated heteroaromatic thiols † Introduction Methylation is one of the simplest and most commonly observed structural modications of biomacromolecules to regulate their functions. In proteins, posttranslational methylation occurs at several amino acid residues such as lysine, arginine, and histidine. 1,2 Methylation at a heteroatom in a side chain supplies the amino acid residue with a cationic charge to alter the electronic characteristics at the peripheral and even distantly positioned domains, upon which the protein changes its conformation, localization in the cell, and interactions with partner molecules and substrates. Chaperones, proteins that assist protein folding, are known to be regulated by methylation. Hsp90, for example, is a highly conserved molecular chaperone in the cytoplasm of eukaryotic cells, and its C-terminal domain can be methylated by a methyltransferase. [3][4][5] The methylation leads to a conformational change and enhances interaction with a client protein.
Such "methylation-regulated" functional control inspired us to design biomimetic synthetic folding promotors responsive to methylation.
Folding is an essential reaction of proteins to express their bioactivities by forming native conformations (N). 6,7 Among the folding reactions, oxidative protein folding is a process that includes disulde (SS) bond formation between cysteine residues in the polypeptide chain during the transition from the reduced and unfolded state (R) to N under the thermodynamic or kinetic control (Fig. 1). [8][9][10][11][12][13][14] An R-form polypeptide chain possessing multiple cysteine residues can proceed through several pathways of folding. One is a route that can form N by the straightforward reaction with an oxidant (RS-SR, route 1 in Fig. 1), while the other routes include the formation of a nonnative form (NonN) with SS bonds between non-native cysteine pairs (route 2). The subsequent SS bond isomerization process allows for the conversion of NonN to N while gaining increased thermodynamic stability by cleavage and reformation of SS bonds by inter-and intramolecular nucleophilic attacks of thiolate anions (steps 2-4 in route 2). In cells, redox-active enzymes belonging to the protein disulde isomerase (PDI) family assist the oxidative protein folding by facilitating the formation and isomerization of SS bonds in the client proteins. [15][16][17][18] In PDI, N-and C-terminal thioredoxin-(Trx-) like domains contain redox-functional active centers with the sequence CXHC. 19 One of the notable properties of the CXHC motif is the low pK a of the thiol (SH) groups. The high acidity of the SH groups allows for efficient production of thiolate (S − ) species reactive for SS-bond isomerization in physiological conditions. The imidazolyl group, an aromatic base, in the histidine residue of the CPHC motif of prokaryotic disulde isomerase plays a critical role in increasing the acidity of SH groups in the neighboring Cys residues. 20,21 Inspired by the active sites, synthetic mimics of disulde isomerases have been developed based on redox-active small molecules. Thiols or selenols conjugated with basic units are an established molecular structure as effective folding promotors. [22][23][24][25][26][27][28][29] For example, glutathione (GSH) is known to promote oxidative protein folding, and the replacement of its glutamate residue with arginine leads to a signicant acceleration of the folding reaction. 30 A conjugate between a diselenide and a basic amino acid residue such as histidine is effective for the assistance of protein folding. 31 It has been also reported that thiols bearing an amino or a guanidyl group prompt oxidative folding. 32,33 Importantly, in these previous examples, the synthetic folding-promoting thiols were added in excess amounts relative to the client proteins, likely due to insufficient disulde bond isomerization and anti-aggregation activities.
Although developing small folding promotors functioning by enzymatic or stoichiometric loading is advantageous in industrial applications of protein synthesis and biological applications for redox-based cellular controls, effective molecular design of small thiol compounds with sufficient activities for minimum loading has not been reported. In this study, inspired by the biological methylation-triggered controls of protein activities, we demonstrate the remarkable acceleration of oxidative protein folding facilitated by N-methylation of heteroaromatic thiols with semi-enzymatic efficiencies.
By monitoring the absorption intensity at 240 nm corresponding to thiolate anions at variable pH, pK a values of the thiol groups of PySHs and their methylated forms were characterized (Table 1, Fig. S1 in ESI †). Interestingly, pK a values of PySHs were commonly lower than that of GSH (9.17), 34 likely due to their thiol-base conjugated structures, and pPySH showed the lowest pK a value (oPySH: 9.04; mPySH: 9.05; pPySH: 8.68). Of importance, N-methylation further lowered the pK a values of the thiol groups (oMePySH: 6.23; mMePySH: 7.64; pMePySH: 7.34). Redox potential E°′ is an indicator of the reducing power of a thiol group; higher E°′ indicates weaker reductant. In other words, the corresponding disulde form possesses a higher oxidizing capability. PySHs and MePySHs, except oPySH, commonly showed higher E°′ values than GSH ( Table 1, Fig. S2 in ESI †). 35 The E°′ values of MePySHs were also higher than those of the corresponding PySHs. Fig. 1 Schematic illustration of oxidative folding of a polypeptide chain forming two disulfide bonds. Orange and yellow circles represent cysteine residues that form disulfide bonds between the same color pairs, and white circles represent amino acid residues other than cysteine. Route 1: oxidation reaction directly from a reduced and unfolded form (R) to the native form (N). Route 2: oxidation reaction (step 1) followed by disulfide bond isomerization (steps 2-4) from R to N via non-native form (NonN).

Folding promotion of ribonuclease A by PySHs and MePySHs
To study the folding promotion effects of the PySHs and their methylated forms, SS bond formation and enzymatic activity recovery of reduced and unfolded ribonuclease (RNase) A were monitored. RNase A is a representative protein used in oxidative folding assays, possessing four disulde bonds between cysteine residues at C26-C84, C40-C95, C58-C110, and C65-C72. 10 Fig. 3C). At 30 min incubation, RNase A showed a stronger band of FOS in the presence of oPySH than GSH in the SDS-PAGE analysis; therefore, it is indicated that the oPySH/GSSG system has a higher acceleration effect of SS bond formation than the GSH/GSSG system. Based on the electrophoretic assays ( Fig. 3B-E), quantitative time-course proles of RNase A FOS formation indicate that the oPySH/GSSG system showed the fastest SSbond formation and the reaction rate decreased in the order of the pPySH/GSSG and mPySH/GSSG systems (Fig. 3I).  Interestingly, in the recovery rate of the enzymatic activity of RNase A to monitor the formation of its native form, the pPySH/ GSSG system showed the highest efficiency among the promethylated PySHs (Fig. 3J). By N-methylation of PySHs, characteristics of these compounds were emphasized more sharply. The oMePySH/ GSSG system converted 67% of RNase A to FOS within 10 min incubation, and the conversion was further enhanced to 80% by extending the incubation longer than 30 min (Fig. 3F and K). The FOS formation rate became slower in the order of oMe-PySH, pMePySH, and mMePySH in common with their promethylated forms (Fig. 3F-H and K). In the enzymatic activity monitoring, on the other hand, the addition of mMePySH and pMePySH showed signicantly faster recovery rates than that of oMePySH (Fig. 3L). The rate constants for RNase A refolding were determined to be k GSH = 2.7 × 10 −3 min −1 , k oMePySH = 7.7 × 10 −3 min −1 , and k pMePySH = 8.5 × 10 −3 min −1 (calculated by IGOR Pro), i.e., the reaction rate using pMePySH was more than three times faster than that using the typical glutathione system. Given that the enzymatic activity exclusively originated from the native form, the RNase A refolding in the presence of oMePySH should be highly error-prone, possibly producing oxidized non-native forms. Thus, N-methylation of pyridinylmethanethiols commonly accelerated the FOS formation of RNase A compared to the corresponding pro-methylated forms, and mMePySH and pMePySH demonstrated efficient refolding promotion of RNase A to the native form.

Folding promotion of bovine pancreatic trypsin inhibitor by PySHs and MePySHs
To verify the generality of the folding acceleration by the Nmethylation of pyridinylmethanethiols, the folding reaction of bovine pancreatic trypsin inhibitor (BPTI) was investigated in the presence of GSSG as an oxidant and the synthesized thiol compounds ([BPTI] = 30 mM; [GSSG] = 0.20 mM; [thiol] = 1.0 mM). N-BPTI contains three disulde bonds at C5-C55, C14-C38, and C30-C51. The folding process of BPTI from its R-form proceeds via quasi-native intermediates, i.e., N ′ , N*, and N SH SH (Fig. 4A), 39 that can be monitored quantitatively by reversedphase high performance liquid chromatography (HPLC), useful for the time-course analysis of the folding progress. The HPLC analysis showed that the fraction of R-BPTI disappeared within 10 min of the incubation in the presence of oPySH/GSSG, and in the meantime, the fractions corresponding to the intermediates and N-BPTI appeared and intensied to reach 38% yield of N-BPTI aer 60 min incubation ( Fig. 4B and H). Interestingly, mPySH/GSSG and pPySH/GSSG systems afforded increased yields of N-BPTI aer 60 min incubation (mPySH/GSSG: 48%; pPySH/GSSG: 51%; Fig. 4C, D and H). Importantly, the Nmethylated thiols prompted the folding of BPTI more efficiently. In the oMePySH/GSSG system, the fraction of R-BPTI disappeared within 1 min incubation, and notably, formation of N-BPTI was visualized aer the initial 1 min incubation. The area of the N-BPTI fraction increased upon extension of the incubation time to provide 53% yield aer 60 min incubation ( Fig. 4E and I). Further enhanced BPTI folding promotion was demonstrated by the pMePySH/GSSG system. R-BPTI was rapidly oxidized within 1 min incubation, at which the fraction of N-BPTI appeared. By extending the incubation up to 60 min, the yield of N-BPTI increased to 71% (Fig. 4G and I).

Discussion of chemical properties of PySHs and MePySHs on the folding process
As demonstrated in the RNase A and BPTI folding assays, the Nmethylation signicantly enhanced the yields of the nativestructure formation. HPLC analysis indicated that, in the preincubated mixture of pMePySH and GSSG, pMePySS and pMePyS-SG were generated (Fig. S3 †). It is likely that pMePySS and pMePyS-SG generated in situ react with a protein as oxidants to facilitate the disulde-bond formation, therefore the oxidation of the proteins is accelerated (step 1 in Fig. 1). The added thiol compound, pMePySH, accelerates the disuldebond isomerization by an intermolecular nucleophilic attack to form pMePyS-Cys and Cys exposing a free thiol group, and the regenerated Cys can attack another Cys-Cys by an intramolecular nucleophilic reaction (steps 2-4 in Fig. 1). The disulde-bond isomerization should allow for conformational changes of the protein to gain increased thermodynamic stability to lead to the folding into its native structure. Therefore, higher oxidizability and nucleophilicity of pMePySS and pMePySH, respectively, accelerate the overall oxidization of the client proteins to afford FOS and enhances the shuffling efficiency to promote the folding to the native form.
In the RNase A folding assays, disulde-bond formation to afford FOS proceeded faster in the presence of MePySHs/GSSG than PySHs/GSSG ( Fig. 3I and K), where MePySS and PySS would be presumably generated in the preincubated mixture for acting as oxidants. The rapid disappearance of the R-BPTI fractions within 1 min incubation also demonstrates the faster disulde-bond formation in the presence of MePySHs/ GSSG (Fig. 4E-G). The faster disulde-bond formation by MePySHs than PySHs would be attributed to their higher E°′. The lower pK a of MePySHs than the corresponding PySHs should be advantageous for the nucleophilic attack to the Cys-Cys bonds to facilitate disulde-bond isomerization. Here, it may look contradictory that oPySH showed the fastest disuldebond formation of RNase A despite its signicantly low E°′ value. The fastest disulde bond formation by oPySH could be attributed to the formation of intermolecular disulde bonds between oPySH and cysteine residues of RNase A, which would be stabilized by the reductive property of oPySH. 40 Among MePySHs, it is characteristic that oMePySH propelled the rapid disulde bond formation of RNase A and BPTI. Irrespective of the rapid disulde bond formation, oMePySH showed the lowest efficiency of refolding to the native forms among MePySHs (Fig. 3L and 4I). One critical reason for the low folding promotion efficiency is the formation of non-native and off-pathway intermediates in the presence of oMePySH. Formation of the non-native and off-pathway species is indicated in Fig. 4E, where BPTI showed numerous fractions between the fractions of N* and R aer 1 min incubation and a complex fraction pattern was observed between the N-and N ′ -fractions even aer 60 min incubation. The long retention of the non-native and off-pathway species of BPTI over the 60 min incubation process suggests low SS-bond shuffling capability of oMePySH. The nucleophilicity of the thiol group of oMePySH would be reduced possibly by the steric hindrance with the methyl group close to the thiol group and stabilization of the thiolate anion by the neighboring cationic nitrogen atom. As a result, pMePySH showed superior performance in the promotion of oxidative protein folding to prompt disulde bond formation and afford the native forms in the highest yields. It is likely that the higher E°′ and lower pK a values of pMePySH than mMePySH allowed for the faster disulde bond formation and efficient disulde bond shuffling to provide the native form proteins effectively (Fig. 3L, 4F and G).

Semi-enzymatic folding promotion by pMePySH/pMePySS
In the endoplasmic reticulum where oxidative folding mainly occurs, PDI plays a central role in the assistance of oxidative protein folding. [15][16][17][18] Due to the molecular recognition property, PDI monomer or its dimer binds with a substrate protein to prompt the formation and shuffling of disulde bonds directed to forming the native structures along the client folding pathway. 41 In contrast to such "one-to-one" enzymatic reactions between PDI and substrates, conventional low-molecularweight compounds such as glutathione are commonly used on the mM-order against several mM substrates for protein folding promotion. 41,42 The high performance of pMePySH inspired us to attempt an oxidative protein folding promotion under a more biomimetic condition, i.e., protein folding in the presence of only 1-equivalent of an additive to the number of disulde bonds in a client protein. Surprisingly, even in such a semi-enzymatic condition, highly efficient protein folding promotion could be demonstrated by the N-methylated compound.
For this semi-enzymatic assay, we chose pMePySS, the disulde form of pMePySH, because of its sufficient watersolubility, high oxidizability, and the productivity of pMe-PySH, which showed the highest folding promotion performance by encouraging SS-bond shuffling aer the oxidation reaction. We added 90 mM pMePySS to R-BPTI (30 mM) as the minimum loading for oxidizing the six cysteine residues in BPTI to form three disulde bonds. HPLC analysis indicated the disappearance of R-BPTI and formation of N-BPTI within the initial 5 min incubation, suggesting rapid progress of the oxidation reaction. Aer 60 min incubation, N-BPTI formed in 56% yield, and the yield was further enhanced to 74% aer 180 min incubation ( Fig. 5A and C). In contrast, the oxidation of R-BPTI proceeded signicantly slower over 30 min, and formation of N-BPTI was much less efficient in the presence of oxidized glutathione (GSSG, 90 mM) compared to pMePySS. The HPLC analysis indicated that the yield of N-BPTI was limited to only 38% even aer 180 min incubation (Fig. 5B). As expected, among the constitutional isomers of MePySSs, pMePySS showed the most efficient protein folding promotion in the 1equivalent-loading semi-enzymatic condition ( Fig. 5D and S4 in ESI †). The N-methylated pyridinylmethanethiols also accelerated the folding of RNase A in the semi-enzymatic condition (Fig. S5 in ESI †). Furthermore, to evaluate the practical signicance, we investigated the folding promotion of human insulin, a therapeutic protein, by pMePySS. Insulin comprises two peptide chains (A-and B-chains) with two interchain disulde bridges and one intrachain disulde bond in A-chain; therefore, its folding velocity and efficiency are essentially low. 43 Importantly, under a catalytic condition, pMePySS (200 mM) showed increased efficiency of the chain-coupling of A-and B-chains (200 mM each) of human insulin to afford its native form with a 4.2-fold higher yield relative to GSSG (Fig. S6 and S7 in ESI †). Hence, high activation of thiols for the protein folding promotion was demonstrated by the N-methylation of the pyridinyl group; and particularly pMePySH and its oxidized form promoted and accelerated the oxidative protein folding efficiently even by stoichiometric amount loading.

Conclusions
We have demonstrated the remarkable N-methylation effects of heteroaromatic thiols on the promotion of oxidative protein folding. N-Methylation is a ubiquitous and central posttranslational processing of proteins for their functional regulation including chaperone activities. Although it has been revealed that methylation at a histidine residue is involved in the switching of chaperone functions such as Hsp90, there are few examples of research on the regulation of chaperone function by methylation, and much remains elusive. Despite the universally observed biochemical reactions in nature, N-methylation has hardly been utilized in the design, functionalization and switching of synthetic bioregulatory agents, particularly in the development of folding promotors. Based on this comprehensive study of PySHs including all constitutional isomers and their N-methylated forms revealing the geometrical dependency on the chemical properties, we demonstrated highly efficient folding promotion by pMePySH. Chemical characterizations indicated that N-methylation enhances both acidity and oxidizability of the thiol groups, which likely enabled the remarkable enhancement of the folding reactions by accelerated SS-bond formation and effective shuffling. Using pMePySS, we successfully demonstrated the rst example of the semienzymatic promotion of oxidative protein folding upon 1equivalent loading of the chemical oxidant, which afforded the native form protein in over 70% yield. The development of small chemical compounds that assist and promote protein folding is critically important not only in basic biological studies for the synthesis of natural and mutant proteins but also in industrial applications to produce protein pharmaceuticals such as insulin. Of note, enzymatic dysfunction such as PDI can result in pathological outcomes such as neurodegenerative disease and diabetes. 44 The chemical structure-based design for semienzymatic agents that can replace the enzymes may thus give clues to new approaches for preventing enzyme-related pathogenic protein aggregation. This study will offer a chemical viewpoint of methylation effects on the regulation of enzymatic activities as well as an effective bioinspired molecular design of functional compounds that are useful for the promotion of oxidative protein folding in vitro by minimum loading.

Data availability
Information supporting this article has been uploaded as part of the ESI. † Additional data are available from the authors on reasonable request.

Conflicts of interest
There are no conicts to declare.