Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein.
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.
Hink
M A
MA
MicroSpectroscopy Centre, Department of Biomolecular Sciences and the Laboratory for Monoclonal Antibodies, Wageningen University, 6703 HA Wageningen, The Netherlands.
Griep
R A
RA
Borst
J W
JW
van Hoek
A
A
Eppink
M H
MH
Schots
A
A
Visser
A J
AJ
D000818:Animals; D002473:Cell Wall / Q000276:immunology; D003196:Computer Graphics; D005454:Fluorescence Polarization; D006090:Gram-Negative Bacteria / Q000276:immunology; D049452:Green Fluorescent Proteins; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000276:immunology; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D037401:Scyphozoa; D057127:Single-Chain Antibodies; D013050:Spectrometry, Fluorescence
D007135:Immunoglobulin Variable Region; D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins