Physical characterization of plakophilin 1 reconstituted with and without zinc.
Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual 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 experiments, 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 dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The alpha helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20, w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 x 10-7 cm2.s-1. The molecular mass of 2.44 x 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.
Hofmann
I
I
Division of Cell Biology, German Cancer Research Center, Heidelberg. i.hofmann@dkfz-heidelberg.de
Mücke
N
N
Reed
J
J
Herrmann
H
H
Langowski
J
J
D002942:Circular Dichroism; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008027:Light; D008666:Metalloendopeptidases; D008854:Microscopy, Electron; D008962:Models, Theoretical; D051191:Plakophilins; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013057:Spectrum Analysis; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014461:Ultracentrifugation; D015032:Zinc / Q000378:metabolism*
C495335:PKP1 protein, human; D051191:Plakophilins; D011506:Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D015032:Zinc