Unfolding and conformational studies on bovine adrenodoxin probed by engineered intrinsic tryptophan fluorescence.
An intrinsic steady-state fluorescent system for bovine adrenodoxin has been developed to study the protein structure in solution and the processes involved in protein unfolding. Since mature Adx contains no natural Trp residue as internal probe, all of the aromatic amino acids, tyrosine at position 82 and four phenylalanines at positions 11, 43, 59 and 64, were at each case replaced by tryptophan. The resulting single tryptophan containing mutants kept their biological function compared with the wild type. Molecular modeling studies verify thermal unfolding experiments which point to a dramatically reduced stability caused by steric hindrance only for mutant F59W. Fluorescence spectra, Stern-Volmer quenching constants, and fluorescence energy transfer calculations indicated the analyzed positions to be situated in solution in the same immediate environment as in the crystal structure. Unfolding experiments with Gdn-HCl and time-resolved stopped-flow measurements provide evidence for differential stability and a chronologically ordered unfolding mechanism of the different fluorescence probe positions in the protein.
Hannemann
Frank
F
FR 8.8 Biochemie, Universität des Saarlandes, D-66041 Saarbrücken, Germany.
Bera
Aloke Kumar
AK
Fischer
Birgitta
B
Lisurek
Michael
M
Teuchner
Klaus
K
Bernhardt
Rita
R
D020106:Acrylamide / Q000737:chemistry; D000325:Adrenodoxin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D000325:Adrenodoxin; D020106:Acrylamide; D014364:Tryptophan