Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer.


Abstract

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 > or = 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. Study holds ProTherm entries: 19919, 19920, 19921, 19922, 19923, 19924, 19925, 19926, 19927, 19928, 19929, 19930, 19931, 19932, 19933, 19934, 19935, 19936, 19937, 19938, 19939, 19940, 19941, 19942, 19943, 19944 Extra Details: ligand binding; anions; relative affinity; binding sites

Submission Details

ID: CeutTaDR

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Huus K;Havelund S;Olsen HB;Sigurskjold BW;van de Weert M;Frokjaer S,Biochemistry (2006) Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer. PMID:16548529
Additional Information

Number of data points 45
Proteins Insulin ; Insulin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal details:Additives zinc (),phenol (40 mM), resorcinol (40 mM),, ionic:KSCN: ; Experimental Assay: dHvH details:Additives zinc (),phenol (40 mM), resorcinol (40 mM),, ionic:KSCN: ; Experimental Assay: dHcal details:Additives zinc (),phenol (40 mM),, ionic:KSCN: 10 mM ; Experimental Assay: Tm details:Additives zinc (),phenol (40 mM),, ionic:KSCN: 10 mM ; Experimental Assay: dHvH details:Additives zinc (),phenol (40 mM),, ionic:KSCN: 10 mM ; Experimental Assay: dHcal ionic:4H3N: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: Tm ionic:4H3N: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHvH ionic:4H3N: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHcal ionic:PABA: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: Tm ionic:PABA: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHvH ionic:PABA: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHcal ionic:NaCl: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: Tm ionic:NaCl: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHvH ionic:NaCl: 10 mM, details:Additives zinc (),phenol (40 mM), ; Experimental Assay: dHcal details:Additives zinc (),phenol (40 mM),, ionic:: ; Experimental Assay: Tm details:Additives zinc (),phenol (40 mM),, ionic:: ; Experimental Assay: dHvH details:Additives zinc (),phenol (40 mM),, ionic:: ; Experimental Assay: Tm ionic:KSCN: 10 mM, details:Additives zinc (), ; Experimental Assay: Tm ionic:4H3N: 10 mM, details:Additives zinc (), ; Experimental Assay: Tm ionic:PABA: 10 mM, details:Additives zinc (), ; Experimental Assay: Tm ionic:NaCl: 10 mM, details:Additives zinc (), ; Experimental Assay: Tm details:Additives zinc (),, ionic:: ; Experimental Assay: dHcal details:Additives zinc (), resorcinol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHvH details:Additives zinc (), resorcinol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHcal details:Additives zinc (), phenol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHvH details:Additives zinc (), phenol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHcal details:Additives zinc (), m-cresol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHvH details:Additives zinc (), m-cresol (),, ionic:NaCl: 50 mM ; Experimental Assay: dHcal details:Additives zinc (), resorcinol (),, ionic:: ; Experimental Assay: dHvH details:Additives zinc (), resorcinol (),, ionic:: ; Experimental Assay: dHcal details:Additives zinc (), phenol (),, ionic:: ; Experimental Assay: dHvH details:Additives zinc (), phenol (),, ionic:: ; Experimental Assay: dHcal details:Additives zinc (), m-Cresol (),, ionic:: ; Experimental Assay: dHvH details:Additives zinc (), m-Cresol (),, ionic:: ; Experimental Assay: dHcal details:Additives zinc (),, ionic:NaCl: 50 mM ; Experimental Assay: dHvH details:Additives zinc (),, ionic:NaCl: 50 mM ; Experimental Assay: dHcal details:Additives zinc (),, ionic:: ; Experimental Assay: dHvH details:Additives zinc (),, ionic:: ; Experimental Assay: Tm ionic:: , details:Additives m-cresol (100 mM), ; Experimental Assay: Tm ionic:: , details:Additives resorcinol (100 mM), ; Experimental Assay: Tm ionic:: , details:Additives phenol (100 mM), ; Experimental Assay: Tm ionic:: , details:Additives m-cresol (20 mM), ; Experimental Assay: Tm details:Additives resorcinol (20 mM),, ionic:: ; Experimental Assay: Tm details:Additives phenol (20 mM),, ionic:: ; Experimental Assay: Tm details:Additives , ionic::
Libraries Mutations for sequence A:GIVEQCCTSICSLYQLENYCN/B:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/C:GIVEQCCTSICSLYQLENYCN/D:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/E:GIVEQCCTSICSLYQLENYCN/F:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/G:GIVEQCCTSICSLYQLENYCN/H:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/I:GIVEQCCTSICSLYQLENYCN/J:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/K:GIVEQCCTSICSLYQLENYCN/L:FVNQHLCGSHLVEALYLVCGERGFFYTPKT
Sequence Assay Result Units