Identification and crystallisation of a heat- and protease-stable fragmentof the bacteriophage T4 short tail fibre

Irreversible binding of T-even bacteriophages to Escherichia coli is mediat ed by the short tail fibres, which serve as inextensible stays during DNA i njection. Short tail fibres are exceptionally stable elongated trimers of g ene product 12 (gp12), a 56 kDa protein. The N-terminal region of gp12 is i mportant for phage attachment, the central region forms a long shaft, while a C-terminal globular region is implicated in binding to the bacterial lip opolysaccharide core. When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsi n at 37 degreesC, an N-terminally shortened fragment of 52 kDa resulted. If the protein was incubated at 56 degreesC before trypsin treatment at 37 de greesC, we obtained a stable trimeric fragment of 3 x 33 kDa lacking residu es from both the N- and C-termini. Apparently, the protein unfolds partiall y at 56 degreesC, thereby exposing protease-sensitive sites in the C-termin al region and extra sites in the N-terminal region. Well-diffracting crysta ls of this fragment could be grown. Our results indicate that gp12 carries a stable central region, consisting of the C-terminal part of the shaft and the attached N-terminal half of the globular region. Implications for structure determination of the gp12 prot ein and its folding are discussed.