Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an a-b Parallel Protein Simon Lindhoud 1 , Adrie H. Westphal 1,2 , Jan Willem Borst 1,2 , Carlo P. M. van Mierlo 1 * 1 Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands, 2 Microspectroscopy Centre, Wageningen University, Wageningen, The Netherlands Abstract Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Fo ¨ rster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an a-b parallel protein. Citation: Lindhoud S, Westphal AH, Borst JW, van Mierlo CPM (2012) Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an a-b Parallel Protein. PLoS ONE 7(9): e45746. doi:10.1371/journal.pone.0045746 Editor: Bin Xue, Uni. of South Florida, United States of America Received January 24, 2012; Accepted August 22, 2012; Published September 21, 2012 Copyright: ß 2012 Lindhoud et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Netherlands Organization for Scientific Research (NWO) supported this work (grant number 700.56.002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: carlo.vanmierlo@wur.nl Introduction Folding of proteins to conformations with proper biological activities is of vital importance for all living organisms. To describe protein folding, the concept of a multidimensional energy landscape or folding funnel arose from a combination of experimental data, theory and simulation [1,2,3,4,5]. In this model, proteins descend along a funnel wall describing the free energy of folding, until they reach the native state. Folding energy landscapes usually are rugged and comprise kinetic traps and barriers that pose restrictions on the way to the native state. As a result, partially folded intermediates are formed, which may be on- or off-pathway to the native state. When the intermediate is on- pathway, as is observed for the majority of proteins studied to date, it has native-like topology and is productive for folding. In contrast, when the intermediate is off-pathway, it is trapped in such a manner that the native state cannot be reached without substantial reorganizational events [6]. The resemblance between early kinetic intermediates and molten globules [7,8,9,10] suggests that these molten globules can be considered as models of transient intermediates [6]. Several kinetic studies have revealed involvement of off-pathway interme- diates during protein folding (see e.g. [11,12,13]). The formation of a kinetically trapped off-pathway molten globule increases the likelihood of protein aggregation. Elucidation of the formation and conformation of molten globules offers potential insights into factors responsible for protein misfolding, aggregation, and, potentially, for numerous devastating pathologies [14,15]. Structural characterization of molten globules is hampered by the often transient nature of their existence, their usually relatively low population at equilibrium, and their aggregation at high protein concentrations [16]. One needs techniques that detect these species with high sensitivity, and thus fluorescence spectros- copy and the phenomenon of Fo ¨rster Resonance Energy Transfer are very suitable [17,18,19]. FRET is the distance dependent transfer of electronic excitation energy from a donor fluorophore to an acceptor chromophore through nonradiative dipole-dipole coupling. This phenomenon enables detection of distances between donor and acceptor molecules of typically ,10 nm [17,20,21]. The FRET efficiency (E) strongly depends on the distance (r) between a donor and an acceptor molecule, according to: E~ 1 1z r=R 0 ð Þ 6 ð1Þ with R 0 being the Fo ¨rster distance, i.e., the distance at which the energy transfer efficiency is 50%. Labeling of proteins with bright donor and acceptor dyes strongly facilitates the use of FRET to study protein folding. Here, we employ FRET to study the folding PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45746