title: Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core.
abstract: The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.
authors.affiliation: Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
authors.forename: Edi
authors.initials: E
authors.lastname: Gabellieri
authors.forename: Gijs I
authors.initials: GI
authors.lastname: van Boxel
authors.forename: J Baz
authors.initials: JB
authors.lastname: Jackson
authors.forename: Giovanni B
authors.initials: GB
authors.lastname: Strambini
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D009250:NADP Transhydrogenases / Q000737:chemistry*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011522:Protons; D009243:NAD; D020106:Acrylamide; D009249:NADP; D014364:Tryptophan; D009250:NADP Transhydrogenases; D005990:Glycerol