Protein stability and mutations in the axial methionine loop of a minimal cytochrome c.
The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.
Bartalesi
Ilaria
I
Magnetic Resonance Center, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy.
Bertini
Ivano
I
Di Rocco
Giulia
G
Ranieri
Antonio
A
Rosato
Antonio
A
Vanarotti
Murugendra
M
Vasos
Paul R
PR
Viezzoli
Maria Silvia
MS
D000596:Amino Acids / Q000737:chemistry; D001407:Bacillus / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D019791:Guanidine; D007501:Iron / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D000596:Amino Acids; D008024:Ligands; D014867:Water; D045304:Cytochromes c; D008715:Methionine; D007501:Iron; D019791:Guanidine