W0106

Protein Crystals Grown In Vivo. Nathan Coussens ^‡, Barbara Stay^¥, Kenneth Murphy^‡, James Gray^‡, Jason Telford^£ & S. Ramaswamy^‡§m, ^‡Dept. of Biochemistry, Univ. of Iowa, ^¥Dept. of Biological Sciences, Univ. of Iowa, ^£Dept. of Chemistry, Univ. of Iowa, ^§Dept. of Chemical & Biochemical Engineering, Univ. of Iowa.

Protein crystals were isolated from the midgut of Diploptera Punctata embryos. We collected diffraction data to 1.1Å with these in vivo grown crystals, using syncrotron radiation. These crystals were also dissolved and the protein was co-crystallized with many heavy atoms using the hanging drop method. Currently we are using multiple isomorphous replacement (MIR) methods to obtain phases. We will use the resulting phases to build a model in atomic detail. In addition, we have studied the protein with Circular Dichroism (CD), Differential Scanning Calorimetry (DSC) and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS). The CD spectra from 260 nm to 188 nm, clearly indicates secondary structure and the presence of α-helixes. Our DSC data at pH 9 show two transitions, suggesting multiple domains. However, at pH 4 we see a single peak with the T_m shifted to a higher temperature indicating protein stabilization and suggesting a two-state transition. MS data confirms that the protein is heavily glycosylated. We observe mass differences in the spectra, which correspond to the molecular weights of hexoses, N-acetylhexosamine and sialic acids. Large changes in the mass spectrum of the protein following a digestion with α-mannosidase were observed.