title: Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding.
abstract: Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a 'P-loop' sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.
authors.affiliation: Department of Plant Biology, University of Illinois, Urbana, IL, 61801, USA.
authors.forename: Dafu
authors.initials: D
authors.lastname: Wang
authors.forename: Archie R
authors.initials: AR
authors.lastname: Portis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D014026:Nicotiana / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; C054765:rca protein, plant; D014364:Tryptophan; D000255:Adenosine Triphosphate