PREPARATION OF A PROTEASE RESISTANT DOMAIN OF THE [[alpha]]-HEMOLYSIN MONOMER FOR CRYSTALLIZATION. Y.-D. Mo and J.E. Gouaux, Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL

[[alpha]]-Hemolysin ([[alpha]]HL) is secreted as a water-soluble monomer of 33.4 kDa that self-assembles to form heptameric transmembrane pores. Molecular entities along this pathway are the water-soluble monomer (1), the membrane-bound monomer (2), the heptameric prepore (3) and the fully assembled heptameric channel (4)¹. To elucidate molecular mechanisms for the assembly, we aim to determine high resolution structures of each species using x-ray diffraction techniques. The goal of this work is to determine the structure of the water-soluble monomer. However, the crystallization of wild-type [[alpha]]HL in a monomeric, water soluble form has proven difficult. Therefore, we have focused on partial proteolysis of the [[alpha]]HL monomer to obtain a species suitable for crystallization. Our assumption is that regions of the monomer that are easily removed by proteases under nondenaturing conditions represent flexible entities that are not portions of well-defined secondary or tertiary structure.

Screening and optimization of partial proteolysis conditions have been carried out using trypsin², proteinase K³, elastase, pepsin, papain and subtilisin. In agreement with previous studies, limited proteolysis by trypsin, proteinase K and elastase produced one major protein band of about 17 kDa, indicating that cleavage occurs within the glycine-rich region near the middle of the primary structure. Further proteinase K treatment after cleavage by trypsin generated a major fragment with lower molecular weight than that produced by trypsin or proteinase K alone. The sequence and size of each proteolytic fragment will be determined by amino acid sequencing and high resolution electrospray mass spectrometry. Identification, characterization and purlfication of the major fragment of the trypsin-proteinase K digestion is in progress and further biochemical and crystallographic experiments will be reported.

¹Walker, B., et al. (1992) J. Biol. Chem. 267 21782-86.

²Blomqvist, L et al. (1987) FEBS Lett. 211 127

³Tobkes, N., Wallace, B.A. and Bayley, H. (1985) Biochemistry 24 1915-20.