Green fluorescent protein as a probe of rotational mobility within bacteriophage T4

Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads an d proheads as a probe of the internal environment. Targeting was accomplish ed with internal protein III (IPIII) fusion proteins or capsid targeting se quence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS del IIPIII del GFP], T4[CTS del IIPIII(T)G FP], and T4[CTS del GFP] packaged GFP fusion proteins and processed them at cleavage sites designated del. 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 g ave 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 clo se associations between GFP molecules resulting in homotransfer between flu orophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the conde nsed DNA, and properties of encapsidated viral enzymes. (C) 2000 Published by Elsevier Science B.V. All rights reserved.