Protein stability and mutations in the axial methionine loop of a minimal cytochrome<Emphasis Type="Italic">
c</Emphasis> |
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Authors: | Ilaria?Bartalesi Email author" target="_blank">Ivano?BertiniEmail author Giulia?Di?Rocco Antonio?Ranieri Antonio?Rosato Murugendra?Vanarotti Paul?R?Vasos Maria?Silvia?Viezzoli |
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Institution: | (1) Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy;(2) Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy;(3) Department of Chemistry, University of Modena and Reggio Emilia, Via G. Campi 183, 41100 Modena, Italy |
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Abstract: | 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.Abbreviations Bpcytc
soluble fragment of cytochrome c553 from Bacillus pasteurii
- GdmCl
guanidinium chloride
- I75A
Ile75 to Ala mutant
- I75V
Ile75 to Val mutant
- P72A
Pro72 to Ala mutant
- P72G
Pro72 to Gly mutant
- Q68K
Gln75 to Lys mutant
- WT
wild type |
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Keywords: | Cytochrome c NMR spectroscopy Protein unfolding Site-directed mutagenesis Water-protein interaction |
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