proteins STRUCTURE O FUNCTION O BIOINFORMATICS Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly So ´nia S.Leal and Cla ´udio M.Gomes * Instituto Tecnologia Quı ´mica e Biolo ´gica,Universidade Nova de Lisboa, Oeiras, Portugal INTRODUCTION Protein conformations can rangefrom the tightly packed,homogenous nativestateto the unstructured, unfolded form that comprises a very large ensemble of conformations. In between these extremes, more or less structured protein states can be found,which are pre- sumably intermediates of the folding process and encom- passthe so called molten globules. Since theiroriginal descriptions, 1,2 a large number of protein molten globule states has been described and it is now established that these partly folded forms of proteins are characterized by a defined setof common properties. A typicalmolten globule ischaracterized by (i) secondary structure con- tentcomparable to that of the native state; (ii) absence of mostof the tertiary structure; (iii) a largeexposed hydrophobic surface area as a result of a loosely packed hydrophobic core; and (iv) compactness comparable to thatof the native state, with only slightly increased ra- dius (<20%). 3,4 The role of molten globule states in the protein foldingprocessis far from beingconsensual: whereassomemodelspostulatethatallproteinsfold through a common type of intermediate related to mol- ten globules, 5 otherviewsconsiderthatfolding inter- mediatesdependexclusivelyontheproteinprimary sequence and are therefore dissimilar in respect to their structural properties, 3,6 or simply thathere are no on- pathway intermediaries of folding,and thatall apparent Abbreviations: AaFd,Acidianus ambivalens ferredoxin; ANS,1-anilinonaphthalene- 8-sulfonic acid; FRET, fluorescence resonance energy transfer; GdmCl,Guanidi- nium chloride; TCE, 2,2,2-tricloroethanol. Grantsponsor:Fundac¸a ˜o Cie´ncia e Tecnologia (FCT/MCES, Portugal);Grant numbers: POCTI/QUI/37521, POCTI/QUI/45758. *Correspondence to: Cla ´udio M. Gomes,Instituto TecnologiaQuı ´mica e Biolo ´gica.Universidade Nova de Lisboa.,Av. Repu ´blica 127,2780-756 Oeiras, Portugal. E-mail:gomes@itqb.unl.pt Received 3 August 2006;Revised 12 December 2006;Accepted 31 January 2007 Published online 17 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21448 ABSTRACT The biologicalinsertion of iron–sulfur clusters(Fe–S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of non- native protein conformations in iron–sulfur proteins is rele- vant for the understanding of the folding process and cofac- tor assembly. We have investigated the formation of a mol- ten 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 retainsstructural folding and metalcenters. However, upon increasing the tempera- ture,a seriesof successive modifications occurwithin the protein structure: Fe–S disassembly, loss oftertiary contacts and dissociation of the Zn 2þ site,which is simultaneous to alterationson thesecondarystructure. Upon cooling,an apo-ferredoxin state is obtained, with characteristics of a molten globule: compactness identicalto the nativeform; similarsecondarystructureevidenced by far-UV CD; no near-UV CD detected tertiary contacts; and an exposure of the hydrophobic surfaceevidenced by1-anilino naphtha- lene-8-sulfonic acid (ANS) binding. In contrast to the native form,this apo ferredoxin state undergoes reversible thermal and chemicalunfolding.Its conformational stabilitywas investigated by guanidinium chloride denaturation and this state is 1.5 kcalmol 1 destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe–S pocketprobed the conformational dynamicsof the molten globule state: fluorescence quenching, red edge emission shift analysisand resonance energy transfer to bound ANS evi- denceda restrictedmobilityand confinement within a hydrophobic environment. The possiblephysiological rele- vance of molten globule states in Fe–S proteins and the hy- pothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed. Proteins 2007; 68:606–616. V V C 2007 Wiley-Liss, Inc. Key words: protein folding; molten globule; metalloprotein; iron–sulfur centers; thermophile. 606 PROTEINS V V C 2007 W ILEY- LISS,INC.