Structural Consequences of Chromophore Formation and Exploration of Conserved Lid Residues amongst Naturally Occurring Fluorescent Proteins.

Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the ß-sheets.(1) Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

Function and structure of GFP-like proteins in the protein data bank.

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the ß-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in ß-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.