Aromatic Thiol pK_a Effects on the Folding Rate of a Disulfide Containing Protein

Abstract

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.

Chitosan and Thiolated Chitosan

“…Aromatic thiols, for example, have already been shown to increase the folding rate of disulfide-containing proteins more than aliphatic thiols [30,31]. For a pharmaceutical excipient such as thiolated chitosans, the increased reactivity of the thiol group would further increase its beneficial properties over alkyl thiol chitosan and non-thiolated polymers.…”

Synthesis of Aromatic Monothiols and Aromatic Dithiols to Increase the Folding Rate and Yield of Disulfide Containing Proteins

“…Recently, improved redox buffers have been used, such as aliphatic dithiols, synthetic peptides, and aromatic monothiols. 32,[34][35][36][37][38]116 Aliphatic dithiols, e.g. Furthermore, in previous studies by our group, it has been shown that aromatic monothiols (pK a 5.5-6.6) increased the folding rate and the overall yield of native protein compared to standard aliphatic thiols (pK a 8.5-10).…”

“…35 The thiol in its deprotonated form can act as a nucleophile or a leaving group, and thiol in a disulfide can act as a center thiol (Scheme 2). 35 Disulfide bond formation can be inhibited by the burial of reactive groups, such as protein thiol groups, protein disulfide bonds, and mixed disulfide bonds, in a stable tertiary structure. For instance, the burial of the disulfide bonds in stable tertiary structures prevents their reduction and reshuffling.…”

“…With aromatic monothiols, the folding rate of ribonuclease A was 10-23 times faster than with glutathione at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.70. 4,35,117 Additionally, it was also demonstrated that at a high protein concentration (1 mg/mL of lysozyme), aromatic monothiols, 4-mercaptobenzylphosphonic acid (2) and quaternary ammonium salt (3) (pK a 5.5-6), increased the folding rate and yield of lysozyme by 11 times and 20-40%, respectively, at pH 7, and by 7 times and 25%, respectively, at pH 8 in comparison with glutathione (pK a 8.7). 38 By combining the advantageous properties of aromatic monothiols and aliphatic dithiols, I proposed to design aromatic dithiols which will increase the protein folding rate and overall yield of native protein.…”

“…These concentrations of GSSG were selected as our previous results obtained with RNase A and lysozyme used 0.2 mM or 2 mM GSSG. 4,[35][36][37] These folding experiments were then compared directly with 7 mM GSH and 2 mM GSSG. These concentrations of GSH and GSSG were selected for the folding experiments as they gave the best combination of folding rate and yield in our previous study at 0.1 mg/mL lysozyme concntration.…”