A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein.
The scaffolding protein of bacteriophage P22 directs the assembly of an icosahedral procapsid, a metastable shell that is the precursor for DNA packaging. The full-length protein has been shown previously to exist in a monomer-dimer-tetramer equilibrium of elongated and predominantly alpha-helical molecules. Two deletion-mutant fragments of the scaffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichia coli, and purified. Removal of residues 1 to 140 yields a protein that is assembly-active both in vitro and in vivo, while the removal of the C-terminal 11 residues (293 to 303) leads to complete loss of scaffolding activity. Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent dissociation constants Kd(141-303)=640 microM and Kd(141-292)=880 microM. Tetramer formation is not observed for either fragment; thus, the tetramerization domain of the scaffolding subunit resides in the N-terminal portion of the polypeptide chain. Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly alpha-helical fold in each case. Nonetheless, pronounced differences are observed between spectral signatures of the two fragments. Notably, Raman spectra of fragments 141-292 and 141-303 indicate that elimination of residues 293 to 303 results in unfolding of an alpha-helical coat protein "recognition" domain encompassing about 20 to 30 residues. The thermostability of fragment 141-303, monitored over a wide concentration range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) form, while more cooperative unfolding is observed for the dimeric (high concentration) form. A lesser increase in cooperativity upon dimerization is obtained for fragment 141-292. Additionally, the C-terminal recognition domain constitutes the most stable and cooperative unit in the 141-303 fragment. Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows that the C terminus is the only protected segment of the polypeptide chain. On the basis of the measured hydrodynamic and spectroscopic properties, a domain structure is proposed for the scaffolding subunit. The roles of these domains in P22 procapsid assembly are discussed.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.
R
R
Tuma
M H
MH
Parker
P
P
Weigele
L
L
Sampson
Y
Y
Sun
N R
NR
Krishna
S
S
Casjens
G J
GJ
Thomas
P E
PE
Prevelige
D017100:Bacteriophage P22 / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013059:Spectrum Analysis, Raman; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D010446:Peptide Fragments; D015678:Viral Structural Proteins; C111088:scaffolding protein, bacteriophage P22