Factors Controlling Metal-Ion Selectivity in the Binding Sites of Calcium-Binding Proteins. The Metal-Binding Properties of Amide Donors. A Crystallographic and Thermodynamic Study

Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.

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Metal Ion Binding Properties of Perfluorosulfonate Membranes by Laser Excitation Spectroscopy

Applied Spectroscopy ◽

10.1366/0003702884428356 ◽

1988 ◽

Vol 42 (2) ◽

pp. 293-295 ◽

Cited By ~ 2

Author(s):

E. K. L. Wong ◽

G. L. Richmond

Keyword(s):

Metal Binding ◽

Binding Sites ◽

Metal Ion ◽

Laser Excitation ◽

Ion Binding ◽

Metal Ion Binding ◽

Binding Properties ◽

Metal Binding Sites ◽

Fluorescence Measurements ◽

Experimental Parameters

The metal ion binding properties of the perfluorosulfonate membrane Nafion® have been investigated in this study. The experiments involve laser-induced fluorescence measurements of europium (III) ions which are bound to the membrane. By the exploitation of the hypersensitivity of the D → F transitions of europium (III) to the ligand binding environment, the properties of the metal binding sites have been analyzed as a function of various experimental parameters. The spectra and fluorescence lifetime measurements provide evidence for distinct metal binding sites within the polymer, each of which is sensitive to the conditions of the membrane preparation.

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Calcium-binding properties of human erythrocyte calpain

Biochemical Journal ◽

10.1042/bj3250721 ◽

1997 ◽

Vol 325 (3) ◽

pp. 721-726 ◽

Cited By ~ 28

Author(s):

Mauro MICHETTI ◽

Franca SALAMINO ◽

Roberto MINAFRA ◽

Edon MELLONI ◽

Sandro PONTREMOLI

Keyword(s):

Active Site ◽

Human Erythrocyte ◽

Binding Sites ◽

Calcium Binding ◽

Metal Ion ◽

Primary Event ◽

Binding Properties ◽

Physiological Concentration ◽

Physiological Conditions ◽

Sequential Mechanism

The results presented provide more information on the sequential mechanism that promotes the Ca2+-induced activation of human erythrocyte μ-calpain under physiological conditions. The primary event in this process corresponds to the binding of Ca2+ to eight interacting sites, of which there are four in each of the two calpain subunits. Progressive binding of this metal ion is linearly correlated with the dissociation of the proteinase, which reaches completion when all eight binding sites are occupied. The affinity for Ca2+ in the native heterodimeric calpain is increased 2-fold in the isolated 80 kDa catalytic subunit, but it reaches a Kd consistent with the physiological concentration of Ca2+ only in the active autoproteolytically derived 75 kDa form. Binding of Ca2+ in physiological conditions, and thus the formation of the 75 kDa subunit, can occur only in the presence of positive modulators. These are represented by the natural activator protein, found to be a Ca2+-binding protein, and by highly digestible substrates. The former produces a very large increase in the affinity of calpain for Ca2+, and the latter a smaller but still consistent decrease in the Kd of the proteinase for the metal ion. As a result, both dissociation into the constituent subunits and the autoproteolytic conversion of the native 80 kDa subunit into the active 75 kDa form can occur within the physiological fluctuations in Ca2+ concentration. The delay in the expression of the proteolytic activity with respect to Ca2+ binding to native calpain, no longer detectable in the 75 kDa form, can be attributed to a Ca2+-induced functional conformational change, which is correlated with the accessibility of the active site of the enzyme.

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