This title appears in the Scientific Report : 2020 

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates
Cerminara, Michele
Schöne, Antonie / Ritter, Ilona / Gabba, Matteo / Fitter, Jörg (Corresponding author)
Molekulare Biophysik; ICS-5
Biophysical journal, 118 (2020) 3, S. 688-697
Bethesda, Md. Soc. 2020
10.1016/j.bpj.2019.12.006
Journal Article
Functional Macromolecules and Complexes
Get full text
Restricted
OpenAccess
Restricted
OpenAccess
Get full text
Please use the identifier: http://dx.doi.org/10.1016/j.bpj.2019.12.006 in citations.
Please use the identifier: http://hdl.handle.net/2128/24329 in citations.
The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.