Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein

Structural information on intracellular fusions of the green fluorescent pr otein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry, We have investigated the dynamic properties of GFP alone and fused to a singl e chain antibody raised against lipopolysaccharide of the outer cell wall o f Gram-negative bacteria (abbreviated as scFv-GFP), The scFv moiety was fun ctional as was proven in binding assays, which involved the use of both flu orescence correlation spectroscopy observing the binding of scFv-GFP to Gra m-negative bacteria and a surface plasmon resonance cell containing adsorbe d lipopolysaccharide antigen. The rotational motion of scFv-GFP has been in vestigated with time-resolved fluorescence anisotropy. However, the rotatio nal correlation time of scFv-GFP is too short to account for globular rotat ion of the whole protein. This result can only be explained by assuming a f ast hinge motion between the two fused proteins. A modeled structure of scF v-GFP supports this observation.