Replacement of ProB28 by pipecolic acid protects insulin against fibrillation and slows hexamer dissociation

Abstract

This manuscript is dedicated to Professor Mitsuo Sawamoto's outstanding achievements in polymer chemistry and recognizes his recent retirement from 40 years of exceptional service to Kyoto University.Additional supporting information may be found in the online version of this article. K. Y. Fang and S. A. Lieblich contributed equally to this work.

Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin

“…These efforts, which focused on proline analogs known to incorporate well into recombinant proteins in E. coli, 5 illustrated how proline mutagenesis of insulin can be used to tune its biophysical characteristics. 29,30 Here, we demonstrate the efficient incorporation of three new aliphatic proline residues (4R-methylproline, 4R-Me; 4Smethylproline, 4S-Me; and 4-methyleneproline, 4ene; Figure 1c) at position B28 of recombinantly produced insulin; these insulin variants will be referred to as ins-4R-Me, ins-4S-Me, and ins-4ene, respectively. We find that these modifications alter insulin behavior: replacement of ProB28 with 4ene speeds fibril formation, while 4-methylation accelerates hexamer dissociation without affecting stability against physical denaturation.…”

“…Intrigued by the role that ProB28 plays in insulin biophysics, we recently introduced a series of ncPro residues at position B28 of human insulin. , Because mature insulin contains one proline, a residue-specific replacement approach results in site-specific proline replacement without the need for an orthogonal aminoacyl-tRNA synthetase/tRNA pair. These efforts, which focused on proline analogs known to incorporate well into recombinant proteins in E. coli , illustrated how proline mutagenesis of insulin can be used to tune its biophysical characteristics. , …”

Review of the current state of protein aggregation inhibition from a materials chemistry perspective: special focus on polymeric materials

“…[328][329][330] Tirrell et al have shown that insulin denaturation can be prevented by the modifying it via replacement of the proline residue at position 28 (ProB28) with different groups. 331,332 However, this strategy cannot be applied universally for the stabilization of proteins as the modification may result in proteins losing their activity. 328 Another method is to add external additives (excipients) which change the environment around the protein to enable them to retain their native structure even when subjected to severe stress.…”

Polyethylene-glycol-modified zwitterionic polymer assisted protein aggregation arrest and refolding

“…Tirrell et al showed that modifying the insulin protein by replacing the proline residue present at position 28 with another group prevented the protein from undergoing denaturation. 12,13 However, such a strategy is highly protein-specific, and thus, cannot be applied for different types of proteins. 14 Another easier method to protect proteins from denaturation is the addition of excipients such as the small molecules of proline, 15 polyamines, 16 and guanidine, 17 or various polymeric materials, which alter the external environment of the proteins and allow them to retain their native structure even after exposure to physical or chemical stress.…”