CRYSTALLIZATION SEPARATES A DISTINCT SHORT FORM OF A POLLEN ALLERGEN WITH BIOLOGICAL ACTIVITY. Weber W, Betzel Ch, Bufe A

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and asthma bronchiale. In addition to its allergenicity, a ribonuclease activity has recently been attributed to this 29 kDa protein. The recombinant allergen, though highly purified from a bacterial expression system, has been observed to spontaniously convert to a mixture of various forms with molecular sizes between 10 and 29 kDa. Surprisingly, crystals could be grown from such heterogenous preparations. Single crystals, redissolved and analyzed by SDS-PAGE/immunoblot yielded only one distinct low molecular weight protein which was identified by amino acid sequencing as the C-terminal 13 kDa portion of the allergen. Biological assays with solutions from single crystals demonstrated that both allergenicity as well as RNAse activity are associated with this fragment. The preferential crystal growth of the 13 kDa form (as opposed to the full length protein) indicates the compact conformation of that particular portion of the allergen. In this system crystallization functions as a separation technique which contributes to localize the major functional domain within a larger protein.

Two crystals were used to collect low resolution data up to 4.6 Angstrom on a rotating anode generator. From processing 35 images the space group was evaluated to I4122 with cell dimensions of a = 87.72 Å, b = 87.72 Å., c = 59.61 Å.. 5,581 reflections were merged to a final reduced data set containing 568 reflections and a completeness of 98%. Attempts to collect data to higher resolution using synchrotron radiation are in progress.