Green fluorescent protein as a probe of rotational mobility within bacteriophage T4.
Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS/IPIII/GFP], T4[CTS/IPIII(T)GFP], and T4[CTS/GFP] packaged GFP fusion proteins and processed them at cleavage sites designated /. Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII(T)GFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP. Phage, but not proheads, packaged with fusion protein IPIII(T)GFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII(T)GFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201-1503, USA.
J M
JM
Mullaney
R B
RB
Thompson
Z
Z
Gryczynski
L W
LW
Black
D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002213:Capsid / Q000378:metabolism; D015342:DNA Probes; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D015342:DNA Probes; D004279:DNA, Viral; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins