¹Macromolecular Structure Laboratory, ABL-Basic Research Program, NCI Frederick, Cancer and Development Center, Frederick MD, USA,
²Current address: Institute for Molecular Biotechnology, Jena, Germany

Green fluorescent protein (GFP) from Aequorea victoria (Pacific jellyfish) is part of the luminescence system found in many coelenterants. It transforms the blue light produced by aequorin to green light. This is achieved by a remarkable autocatalytically formed chromophore (Ser65-Tyr66-Gly67 form a 4-(4-hydroxyphenyl methylene)-2-imidazolidine-5-one aromatic system) that absorbs light at 395 and 470 nm (major and minor peak, respectively) and emits it at 508 nm. Many mutants which show altered spectra have been characterized. Because of its spectral properties and the ease of expression, this protein is extensively used for a number of applications in basic research and biotechnology. The structure of GFP has recently been determined for the wild type protein and some of its mutants. Those structures show that residues in direct contact with the chromophore are involved in the majority of mutants that have altered spectra. The most drastic changes are caused by mutation of Tyr 66. Only Structures of Y66H mutants have been solved so far.

A series of variants of GFP based on the mutant F64L, that displays superior expression of functional protein, has been constructed. We present here structures of representative mutants with Tyr 66 replaced by all other aromats. All structures are very similar with the average rmsd's of CA's for pairs of structures of only 0.3 A. Deviations greater than 0.8 A in CA occur only on surface loops. Mutations in the hydrophobic core(F64L, V163A) lead to surprisingly small changes, nevertheless they are responsible for dramatic enhancement of folding efficiency. Mutations of residues in contact with the aromatic system of the chromophore (S65C, I167T) influence the hydrogen bonding pattern and hydrophobic contacts of the chromophore. The changes focus on Thr203 and the triad Ser 65 - Glu 222 - Ser 205, all of which are important for the spectral properties. The blue fluorescent mutant (Y66H) displays a structurally very conserved chromophore. Not only do its two aromatic ring systems superimpose well on those in wild type, but additionally the phenolic oxygen of Tyr 66 is replaced by a water molecule with equivalent interactions. The side chains of phenylalanine or tryptophan in position 66 superimpose nicely on those of tyrosine and histidine, respectively. Analysis of the absorption and fluorescence properties of the Tyr 66 mutants suggests, that a solvent accessible residue is involved in changing fluorescence at low pH. His 148 has tentatively been identified as this residue.

The research was sponsored by the National Cancer Institute, DHHS, under contract with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.