Calcium-Binding Characteristics and Conformational Changes in Metastasin, a Member of S-100 Protein Family

E. A. Dukhanina,^1,2 A. S. Dukhanin,³ M. Yu. Lomonosov,⁴ E. M. Lukanidin,^4,5 and G. P. Georgiev^1,4

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia; fax: (7-095) 135-14-05; E-mail: buh@genom-ii.eimb.rssi.ru

²To whom correspondence should be addressed.

³Russian State Medical University, ul. Ostrovityanova 1, Moscow, 117457 Russia; fax: (7-095) 241-04-32.

⁴Institute of Biology of Genes, Russian Academy of Sciences, ul. Vavilova 34/5, Moscow, 117334 Russia; fax: (7-095) 135-41-05.

⁵Danish Cancer Society, Strandboulevarden 49, 7.1, DK-2100, Copenhagen, Denmark.

Submitted November 20, 1996; revision submitted December 23, 1996.

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Investigation of Ca²⁺-binding characteristics of metastasin (Mts-1) by competition with Fluo-3 revealed two types of Ca²⁺-binding sites in Mts-1 with the geometric mean of their dissociation constant (K_d) value of 2.6 µM for the two EF-sites. The Hill coefficient (n_H) is 0.98. A substantial increase in the affinity of Mts-1 for Ca²⁺ and strong cooperative character of binding (K_d = 0.2 µM, n_H = 1.91) is observed in the presence of the target protein p37 isolated from mouse adenocarcinoma cell lines CSML-100 and CSML-0. Two different hydrophobic sites of binding with the fluorescent probe 2-(p-toluidino)naphthalene-6-sulfonate (TNS) per Mts-1 molecule have been determined. The exposure of the hydrophobic binding sites of the first type are shown to be Ca²⁺-dependent and the hydrophobic binding sites of the second type are exposed independently of Ca²⁺ concentration. A decrease in the number of Ca²⁺-dependent hydrophobic centers in the presence of p37 protein was detected by measurements of TNS fluorescence.

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KEY WORDS: S-100 protein, calcium, Mts-1, Fluo-3, target protein.

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