Insulin analog with additional disulfide bond has increased stability and preserved activity
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Insulin analog with additional disulfide bond has increased stability and preserved activity. / Vinther, Tine N.; Norrman, Mathias; Ribel, Ulla; Huus, Kasper; Schlein, Morten; Steensgaard, Dorte B.; Pedersen, Thomas Å.; Pettersson, Ingrid; Ludvigsen, Svend; Kjeldsen, Thomas; Jensen, Knud Jørgen; Hubálek, František.
In: Protein Science, Vol. 22, No. 3, 2013, p. 296-305.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Insulin analog with additional disulfide bond has increased stability and preserved activity
AU - Vinther, Tine N.
AU - Norrman, Mathias
AU - Ribel, Ulla
AU - Huus, Kasper
AU - Schlein, Morten
AU - Steensgaard, Dorte B.
AU - Pedersen, Thomas Å.
AU - Pettersson, Ingrid
AU - Ludvigsen, Svend
AU - Kjeldsen, Thomas
AU - Jensen, Knud Jørgen
AU - Hubálek, František
N1 - Copyright © 2012 The Protein Society.
PY - 2013
Y1 - 2013
N2 - Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.
AB - Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.
KW - Adipocytes
KW - Amino Acid Substitution
KW - Animals
KW - Antigens, CD
KW - Biological Transport
KW - Blood Glucose
KW - Cells, Cultured
KW - Cystine
KW - Dose-Response Relationship, Drug
KW - Drug Stability
KW - Glucose
KW - Humans
KW - Hypoglycemic Agents
KW - Insulin, Regular, Human
KW - Mutant Proteins
KW - Protein Conformation
KW - Protein Stability
KW - Rats
KW - Rats, Mutant Strains
KW - Rats, Wistar
KW - Receptor, Insulin
KW - Recombinant Proteins
KW - Zinc
U2 - 10.1002/pro.2211
DO - 10.1002/pro.2211
M3 - Journal article
C2 - 23281053
VL - 22
SP - 296
EP - 305
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 3
ER -
ID: 99346558