CRYSTALLIZATION OF THE [[alpha]]-HEMOLYSIN MONOMER. G.-Q. Huang*, A. Villa*, S. Cheley#, C. Shustak#, H. Bayley#, and J.E. Gouaux*. #Worcester Found. for Biomedical Research, Shrewsbury MA 01545, *Department of Biochem. & Mol. Biol., University of Chicago, Chicago IL 60637.

[[alpha]]-Hemolysin ([[alpha]]HL) is secreted from Staph. aureus as a water-soluble monomer of 33.4 kDa that binds to erythrocyte cell membranes and creates a heptameric transmembrane pore¹. Here we present crystallization and crystallographic data on a single amino acid mutant that binds to cell membranes but does not form heptamer (H35->W)². We have obtained 20 crystal forms in the presence of various phospholipids; in the absence of phospholipids, crystal growth has been unsuccessful.

PEG and DiC₇PC produce at least seven different crystal forms. One of the crystal forms (I) diffracts to 3.0 Å resolution and has the space group P4₂2₁2 (a=b =219.7 Å, c=191.3 Å). A related form (II) grows using sodium phosphate and different phospholipids, such as DiC₅PC, MonoC₁₄PC, MonoC₈PC, or mixtures of DiC₅P+MonoC₁₄P or DiC₅P+MonoC₈P. I and II have the same space group and similar a cell dimensions. However, the c dimension of form II is about 4% smaller than the c dimension of form I. We estimate that there are 8-10 monomers in the asymmetric unit of form I on the basis of measurement of crystal volume combined with quantitative amino acid analysis³. A series of strong reflections along 00l is indicative of an axis of noncrystallographic symmetry. These data, combined with analysis of self-rotation functions in which there is a strong peak at [[kappa]]=45deg. parallel to c*, implies that there may be an 8-fold axis of rotational or screw-axis noncrystallographic symmetry oriented along the c axis. There are additional weaker peaks in the self- rotation function maps at [[kappa]] = 120deg. and [[kappa]] =180deg.. [[alpha]]HL obtained from dissolved crystals migrates as a monomer as judged by gel filtration in buffer containing DiC₇PC. Since these crystals are grown in the presence of phospholipids, the structure of the protein in these crystal forms may serve as a good model for a membrane-bound conformation of [[alpha]]HL.

¹Gouaux, J.E., Braha, O., Hobaugh, M.R., Song, L., Cheley, S., Shustak, C. And Bayley, H. (1994) Proc. Natl. Acad. Sci. USA 91 12828-31

²Walker, B. And Bayley, H. (1995) Prot. Engineer. 8 491-5.

³Kwong, P., Pound, A. And Hendrickson, W.A. (1994) J. Appl. Cryst. 27 504-9.