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Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly
Author(s) -
Leal Sónia S.,
Gomes Cláudio M.
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21448
Subject(s) - molten globule , ferredoxin , chemistry , protein folding , crystallography , protein tertiary structure , guanidinium chloride , protein secondary structure , native state , quenching (fluorescence) , protein structure , folding (dsp implementation) , denaturation (fissile materials) , circular dichroism , fluorescence , organic chemistry , biochemistry , enzyme , physics , quantum mechanics , electrical engineering , engineering , nuclear chemistry
The biological insertion of iron–sulfur clusters (Fe–S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of nonnative protein conformations in iron–sulfur proteins is relevant for the understanding of the folding process and cofactor assembly. We have investigated the formation of a molten globule state in the [3Fe4S][4Fe4S] ferredoxin from the thermophilic archaeon Acidianus ambivalens (AaFd), which also contains a structural zinc site. Biophysical studies have shown that, at acidic pH, AaFd retains structural folding and metal centers. However, upon increasing the temperature, a series of successive modifications occur within the protein structure: Fe–S disassembly, loss of tertiary contacts and dissociation of the Zn 2+ site, which is simultaneous to alterations on the secondary structure. Upon cooling, an apo‐ferredoxin state is obtained, with characteristics of a molten globule: compactness identical to the native form; similar secondary structure evidenced by far‐UV CD; no near‐UV CD detected tertiary contacts; and an exposure of the hydrophobic surface evidenced by 1‐anilino naphthalene‐8‐sulfonic acid (ANS) binding. In contrast to the native form, this apo ferredoxin state undergoes reversible thermal and chemical unfolding. Its conformational stability was investigated by guanidinium chloride denaturation and this state is ∼1.5 kcal mol −1 destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe–S pocket probed the conformational dynamics of the molten globule state: fluorescence quenching, red edge emission shift analysis and resonance energy transfer to bound ANS evidenced a restricted mobility and confinement within a hydrophobic environment. The possible physiological relevance of molten globule states in Fe–S proteins and the hypothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed. Proteins 2007. © 2007 Wiley‐Liss, Inc.