Unfolding and Conformational Studies on Bovine Adrenodoxin Probed by Engineered Intrinsic Tryptophan Fluorescence

Abstract

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.

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

“…We also determined the reduction potentials of Fdx1 and Fdx2 to be −267 AE 5 mV and −342 AE 5 mV, respectively (22 (Fig. 2A).…”

Label‐Free Microscale Thermophoresis Discriminates Sites and Affinity of Protein–Ligand Binding

“…If insufficient Trp residues are incorporated, it is possible to introduce Trp by mutation. A conservative exchange of another aromatic amino acid for Trp often does not affect the molecular properties and protein function [24]. Alternatively, labeling and standard MST can be chosen, which typically can be used for lower protein concentrations and thus for the exact determination of affinities in the region of K D <1 n m [9].…”

Functional Characterization of Fdx1: Evidence for an Evolutionary Relationship between P450-Type and ISC-Type Ferredoxins

“…16 Purification of bovine Adx and AdR from recombinant E. coli was also done as described before. 49,50 Isolation of CYP11A1 from bovine adrenal glands was done according to the protocol of Omura and Sato. 51 So ce56 IscU was purified as apoprotein.…”