Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

Abstract

Structural information on intracellular fusions of the green fluorescent protein (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 single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.The green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria has received widespread utilization as a natural fluorescent marker for gene expression, localization of gene products (1-5), and identification of protein interaction and function. GFP is a protein consisting of 238 amino acids with a molecular mass of 27 kDa and has the shape of a cylinder with a length of 4.2 nm and diameter of 2.4 nm. The chemical structure of the hexapeptide chromophore has been elucidated (6). The intrinsic fluorophore is a p-hydroxybenzylidene-imidazolidine derivative formed by a covalent modification of the sequence Ser 65 (or Thr 65 in enhanced GFP), Tyr 66 , and Gly 67 in the hexapeptide. A comprehensive review on GFP has been published (7). The crystal structure of GFP and enhanced GFP has been solved and showed the hexapeptide to be part of a central helix inside a 11-stranded ␤-barrel (8 -11).Genetic fusions of a variety of proteins with GFP have been used in numerous studies on gene or protein function. In a sense it is miraculous that in most fusion proteins GFP is functional. In many other fusion proteins, the protein used as a reporter does often not fold well, resulting in aggregates or inclusion bodies of the entire fusion protein. Sometimes the causes of aggregation can be attributed to certain (clusters of) amino acids such as hydrophobic clusters of amino acids that become solvent exposed (12). To obtain a better picture why these phenomena do not occur in GFP fusion proteins, we have investigated the behavior of the GFP moiety in fusion proteins and emphasized the motional properties. Thereto, we fused enhanced GFP with a single chain Fv fragment raised against the lipopolysaccharide (LPS) of a Gram-negative bacterium. Because the single chain antibody was linked to the N-terminal residue of GFP, the fusion protein is abbreviated as scFv-GFP. Here we report information relevant for the dynamics of GFP fusion proteins used to monitor pro...

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