Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre.
Irreversible binding of T-even bacteriophages to Escherichia coli is mediated by the short tail fibres, which serve as inextensible stays during DNA injection. Short tail fibres are exceptionally stable elongated trimers of gene product 12 (gp12), a 56 kDa protein. The N-terminal region of gp12 is important for phage attachment, the central region forms a long shaft, while a C-terminal globular region is implicated in binding to the bacterial lipopolysaccharide core. When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsin at 37 degrees C, an N-terminally shortened fragment of 52 kDa resulted. If the protein was incubated at 56 degrees C before trypsin treatment at 37 degrees C, we obtained a stable trimeric fragment of 3 x 33 kDa lacking residues from both the N- and C-termini. Apparently, the protein unfolds partially at 56 degrees C, thereby exposing protease-sensitive sites in the C-terminal region and extra sites in the N-terminal region. Well-diffracting crystals 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 protein and its folding are discussed.
van Raaij
M J
MJ
European Molecular Biology Laboratory, Grenoble Outstation, France.
Schoehn
G
G
Jaquinod
M
M
Ashman
K
K
Burda
M R
MR
Miller
S
S
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry; D015678:Viral Structural Proteins / Q000737:chemistry*
D010446:Peptide Fragments; D015678:Viral Structural Proteins; C080832:gp12 protein, Enterobacteria phage T4; D002918:Chymotrypsin; D014357:Trypsin