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

Study Summary

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

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BZV 1998-11-04T00:00:00+0000 0 [D-ALAB26]-DES(B27-B30)-INSULIN-B26-AMIDE A SUPERPOTENT SINGLE-REPLACEMENT INSULIN ANALOGUE, NMR, MINIMIZED AVERAGE STRUCTURE
1BZV 1998-11-04T00:00:00+0000 0 [D-ALAB26]-DES(B27-B30)-INSULIN-B26-AMIDE A SUPERPOTENT SINGLE-REPLACEMENT INSULIN ANALOGUE, NMR, MINIMIZED AVERAGE STRUCTURE
3WS3 2014-02-28T00:00:00+0000 2.33 Crystal Structure of H-2D in complex with an insulin derived peptide
6CE7 2018-02-11T00:00:00+0000 7.4 Insulin Receptor ectodomain in complex with one insulin molecule
6CE9 2018-02-11T00:00:00+0000 4.3 Insulin Receptor ectodomain in complex with two insulin molecules
6CEB 2018-02-11T00:00:00+0000 4.7 Insulin Receptor ectodomain in complex with two insulin molecules - C1 symmetry
1APH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1APH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1BPH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1BPH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
577.8 B,D,F,H,J,L Insulin P67970 INS_CHICK
1149.6000000000001 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01323 INS2_RAT
1149.6000000000001 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01326 INS2_MOUSE
578.4 B,D,F,H,J,L Insulin P51463 INS_SELRF
578.4 B,D,F,H,J,L Insulin P68243 INS_CAIMO
578.4 B,D,F,H,J,L Insulin P68245 INS_ANSAN
578.4 B,D,F,H,J,L Insulin P01333 INS_ANAPL
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P18109 INS_DIDVI
559.8 B,D,F,H,J,L Insulin P67971 INS_SAISC
559.8 B,D,F,H,J,L Insulin P67972 INS_AOTTR
1122.6 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01320 INS_CAMDR
1131.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01324 INS_ACOCA
600.0 B,D,F,H,J,L Insulin P01318 INS_SHEEP
1171.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q62587 INS_PSAOB
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01315 INS_PIG
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P67974 INS_PHYMC
1171.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01310 INS_HORSE
600.0 B,D,F,H,J,L Insulin P06306 INS_FELCA
600.0 B,D,F,H,J,L Insulin P01319 INS_CAPHI
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01321 INS_CANLF
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01317 INS_BOVIN
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P67973 INS_BALPH
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01314 INS_BALBO
1180.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01311 INS_RABIT
1180.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q91XI3 INS_ICTTR
1151.4000000000003 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01313 INS_CRILO
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q8HXV2 INS_PONPY
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30410 INS_PANTR
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30406 INS_MACFA
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01308 INS_HUMAN
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q6YK33 INS_GORGO
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01316 INS_ELEMA
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30407 INS_CHLAE
600.0 B,D,F,H,J,L Insulin F8WCM5 INSR2_HUMAN
543.0 A,C,E,G,I,K Insulin P01327 INS_CHICH
571.2 A,C,E,G,I,K Insulin P01322 INS1_RAT
571.2 A,C,E,G,I,K Insulin P01325 INS1_MOUSE
547.8 B,D,F,H,J,L Insulin P01338 INS2_BATSP
550.2 B,D,F,H,J,L Insulin P68987 INS_PETMA
550.2 B,D,F,H,J,L Insulin P68988 INS_LAMFL
555.6 B,D,F,H,J,L Insulin C0HJI7 INS1_HUSDA
553.8 B,D,F,H,J,L Insulin C0HJI3 INS2_KATPE
553.8 B,D,F,H,J,L Insulin P09476 INS_ATRSP
553.8 B,D,F,H,J,L Insulin Q9W7R2 INS_VERMO
553.8 B,D,F,H,J,L Insulin P09477 INS_PLAFE
553.8 B,D,F,H,J,L Insulin P04667 INS_ONCKE
555.6 B,D,F,H,J,L Insulin P01340 INS_KATPE
558.6 B,D,F,H,J,L Insulin P83770 INSL_VIGUN
555.6 B,D,F,H,J,L Insulin P12707 INS2_XENLA
577.2 B,D,F,H,J,L Insulin C0HJI8 INS2_HUSDA
579.6 B,D,F,H,J,L Insulin P21563 INS_RODSP
600.0 B,D,F,H,J,L Insulin P81423 INS_ACIGU
557.4 B,D,F,H,J,L Insulin P12706 INS1_XENLA
577.8 B,D,F,H,J,L Insulin P69048 INS_TRASC
577.8 B,D,F,H,J,L Insulin P69047 INS_TRADO
577.8 B,D,F,H,J,L Insulin P67969 INS_STRCA
577.8 B,D,F,H,J,L Insulin P67968 INS_MELGA