title: Origins of the Intermediate Spectral Form in M100 Mutants of Photoactive Yellow Protein.
abstract: Numerous single-site mutants of photoactive yellow protein (PYP) from Halorhodospira halophila and as well as PYP homologs from other species exhibit a shoulder on the short wavelength side of the absorbance maximum in their dark-adapted states. The structural basis for the occurrence of this shoulder, called the "intermediate spectral form," has only been investigated in detail for the Y42F mutation. Here we explore the structural basis for occurrence of the intermediate spectral form in a M121E derivative of a circularly permuted H. halophila PYP (M121E-cPYP). The M121 site in M121E-cPYP corresponds to the M100 site in wild-type H. halophila PYP. High-resolution NMR measurements with a salt-tolerant cryoprobe enabled identification of those residues directly affected by increasing concentrations of ammonium chloride, a salt that greatly enhances the fraction of the intermediate spectra form. Residues in the surface loop containing the M121E (M100E) mutation were found to be affected by ammonium chloride as well as a discrete set of residues that link this surface loop to the buried hydroxyl group of the chromophore via a hydrogen bond network. Localized changes in the conformational dynamics of a surface loop can thereby produce structural rearrangements near the buried hydroxyl group chromophore while leaving the large majority of residues in the protein unaffected.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, ON, Canada.
authors.forename: George Andrew
authors.initials: GA
authors.lastname: Woolley
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria