Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer
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Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer. / Huus, Kasper; Havelund, Svend; Olsen, Helle B; Sigurskjold, Bent W; van de Weert, Marco; Frokjaer, Sven.
In: Biochemistry, Vol. 45, No. 12, 28.03.2006, p. 4014-4024.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer
AU - Huus, Kasper
AU - Havelund, Svend
AU - Olsen, Helle B
AU - Sigurskjold, Bent W
AU - van de Weert, Marco
AU - Frokjaer, Sven
PY - 2006/3/28
Y1 - 2006/3/28
N2 - The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate = 4-hydroxy-3-nitrobenzoate p-aminobenzoate chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.
AB - The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate = 4-hydroxy-3-nitrobenzoate p-aminobenzoate chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.
KW - Allosteric Regulation
KW - Biopolymers
KW - Calorimetry, Differential Scanning
KW - Humans
KW - Insulin
KW - Ligands
KW - Phenols
KW - Spectrophotometry, Ultraviolet
KW - Zinc
U2 - 10.1021/bi0524520
DO - 10.1021/bi0524520
M3 - Journal article
C2 - 16548529
VL - 45
SP - 4014
EP - 4024
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 12
ER -
ID: 1100869