Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin, and miniproinsulin.
The chromophoric divalent metal ion chelators 4-(2-pyridylazo)resorcinol (PAR) and 2,2',2"-terpyridine (terpy) are used as kinetic and spectroscopic probes to investigate in solution the SCN- -induced conformational transformations of the insulin, proinsulin, and miniproinsulin hexamers (miniproinsulin is a proinsulin analogue wherein the C-chain is replaced by a dipeptide cross-link between Gly-A1 and Ala-B30). Herein we designate the 2Zn and 4Zn crystal forms of the hexamer as the T6 and T3R3 conformations, respectively. For all three proteins, addition of SCN- reduces the rate of sequestering and removal of zinc ion by chelator. The effect of SCN- on the rate of this process saturates at the same concentration (30 mM) known to induce the T6 to T3R3 transformation in the insulin crystal. Under both T6 and T3R3 conditions, the critical stoichiometry for high-affinity interaction between Zn2+ and each of the three proteins is shown to be 2 mol of Zn2+/mol of protein hexamer. Consequently, we confirm the finding that off-axial coordination of Zn2+ via His-B10 and His-B5 residues is of minor importance for the SCN- -induced conformation change in solution [Renscheidt, H., Strassburger, W., Glatter, U., Wollmer, A., Dodson, G. G., & Mercola, D. A. (1984) Eur. J. Biochem. 142, 7-14]. Under T6 conditions, the kinetics of the reactions between insulin, proinsulin, and miniproinsulin and a variable excess of terpy are similar and biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)
Kaarsholm
N C
NC
Department of Biochemistry, University of California, Riverside 92521.
Ko
H C
HC
Dunn
M F
MF
D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D015394:Molecular Structure; D011384:Proinsulin / Q000378:metabolism*; D012996:Solutions; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D007328:Insulin; D012996:Solutions; C060793:miniproinsulin; D011384:Proinsulin; D015032:Zinc