Physical characterization of plakophilin 1 reconstituted with and without zinc

Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is char acterized by the presence of a conserved 42-amino-acid motif. Despite indiv idual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection ex periments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynami c light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The alpha helix content of P KP1 was considerably higher when reconstituted with zinc than without. By a tomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly assoc iated with one molecule of wild-type PKP1. The zinc-reconstituted protein f ormed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electr on microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s(20,w)) for zinc-containing PKP1 wa s 41S and its diffusion coefficient, as obtained by dynamic light scatterin g, 1.48 x 10(-7) cm(2).s(-1). The molecular mass of 2.44 x 10(6) obtained f rom s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amoun t of zinc, sedimented with 4.6S, and was present in monomeric form as deter mined by sedimentation equilibrium centrifugation.