Analysis of Interhexameric Contact Regions in Two Dodecameric Hemocyanins at Subdohain Resolution by Combination of Data from Electron Microscopy, X-Ray Diffraction and Amino-Acid Sequence Studies

Abstract

Hemocyanin is a biological macromolecule which occurs freely dissolved in the hemolymph of certain invertebrates. The function of this copper containing protein is the transport of oxygen through the organism. In fulfilling this task hemocyanin has developed a similar mechanism as hemoglobin in binding oxygen reversibly and cooperatively. The hemocyanin of arthropods consists of one or more hexamers of subunits with a molecular weight of approx. 75 000. Depending on the species 3-15 types of monomeric subunits occur, which differ in amino-acid composition and in their oxygen binding properties. Each type of subunit fulfills a specific role in the architecture of that hemocyanin. In nature arthropodan hemocyanin is found as a one-hexameric, two-hexameric (dodecameric), four-hexameric or eight-hexameric molecular assembly depending on the species. In this work we focus on the difference in organization of the hexamers in the dodecamer of two different species i.e. the tarantula Eurypelma californicum (a chelicerate) and the crab Cancer pagurus (a crustacean). Eurypelma hemocyanin is made from 7 different subunits called a - g, whereas Cancer hemocyanin consists of 3 subunit types termed α, β, and γ .By image analysis of electron micrographs of the two-hexameric half hemocyanin molecules from Eurypelma and the two-hexameric whole hemocyanin molecules from Cancer, computer averaged projections of these dodecamers were obtained as shown in fig. 1. They differ clearly in their interhexameric contacts. To analyse this difference in more detail these projections were used as a reference in a simulation procedure.