STRUCTURAL STUDY OF PHOTINUS PYRALIS FIREFLY LUCIFERASE USING FLUORESCENCE

摘要

Firefly luciferase (FFL) catalyses the oxidation of a heterocyclic molecule called luciferin (LH_2) in the presence of ATP and oxygen. Reaction proceeds through the formation of a ternary complex followed by an adenylation step in which an intermediate product luciferyl-adenylate is formed. Different observations suggest that FFL undergoes significant conformational changes in the process of the bioluminescent reaction. The crystal structure of FFL without bound substrates was shown to consist of a large N-terminal domain and a small C-terminal domain separated by a wide solvent filled cleft.2 The active centre is considered to be located on the inner surface of the cleft. The conformation of FFL without bound substrates will be further referred to as "opened". All attempts to obtain crystals of FFL with the bound substrates and to resolve its crystal structure by X-ray analysis have failed so far. So there is no direct evidence for the conformational changes. On the other hand it was shown that the crystal structure of phenylalanine-activating subunit of gramicidin synthetase 1, the only FFL homologue with resolved crystal structure, in a complex with phenylalanine, Mg~(2+) and AMP is remarkably similar in size and shape to the FFL "opened" conformation but the C-terminal domain is significantly rotated and approaches the N-terminal domain. It suggests that binding of LH_2 and ATP in the active centre of FFL causes a similar conformational change in its structure which will be further referred to as "closed". Tryptophan (Trp) residue can be used as a sensitive intrinsic fluorescence probe of protein conformational changes, so a catalytically active tryptophan-free mutant (TF) of FFL has been constructed by subsequent site-directed mutagenesis of two naturally occurring Trp residues in positions 417 and 426 (Fig. 1). This has allowed to construct a number of FFL mutants containing single Trp residue in different positions predicted to be sensitive to conformational changes in FFL caused by binding LH_2 and ATP.