4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

Abstract

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

Profiling Insulin Oligomeric States by 1H NMR Spectroscopy for Formulation Development of Ultra-Rapid-Acting Insulin

“…This could be due to complete dissociation of the hexamer, thus no difference in measured dissociation rate, and the broad distribution reporting on the tetramer, dimer, and possibly monomer equilibrium. These observations above are similar to previous reports on noncanonical amino acid-modified insulins, 15 where mutations destabilized insulin dimers but had no effect on hexamer dissociation or vice versa, confirming again the significant potential of these NMR techniques in the solution characterization of insulin oligomeric states.…”

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

“…1–5 Effective exploitation of ncAAs enhances our understanding of basic biology 6–8 and provides opportunities for engineering new classes of materials 9 and biological therapeutics. 2, 10–12 Many of these applications require high efficiency, high fidelity ncAA incorporation and subsequent careful evaluation of such events. The use of mass spectrometry-based characterizations offers the highest level of rigor, 9, 13 but lacks the throughput needed for initial screening and characterization.…”

4‐Naphthylmethyl Proline Forms a Channel Structure

“…This orientation gives rise to smaller channels (Φ = 4.05 Å) that are flanked by the naphthyl groups of three molecules within the same plane (Figure 2,d). 3 The naphthyl moieties of adjacent molecules are in proximity to each other (3.70 Å) as well as to the CHgroups at C(β) and C(γ) of the proline rings (3.60 Å and 3.63 Å), distances indicative of weak π-π and CH-π interactions. [20] The naphthyl groups are not parallel to the z-axis but tilted at an angle α = 61.3°.…”

“…Proline, the only proteinogenic amino acid with a cyclic backbone, is used both by Nature and scientists to control the conformation of peptides and proteins. [1][2][3][4][5] However, proline's unique structural features manifest themselves not only when incorporated into polypeptide chains. In fact, the architecture of monomeric proline crystal structures differs from those of all other amino acids.…”

“…CCDC-1846337 3. The radius of the small channel was defined by the distance between the closest hydrogen atom of the naphthyl ring to the C 3 rotation axis.…”