title: Extensive formation of off-pathway species during folding of an alpha-beta parallel protein is due to docking of (non)native structure elements in unfolded molecules.
abstract: Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent alpha-beta parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast, at 3.4 M denaturant, secondary shifts and (1)H-(15)N relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form alpha-helices, which are populated about 10% of the time, as confirmed by far-UV CD data. One region of unfolded apoflavodoxin adopts non-native structure. Of the alpha-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes beta-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter alpha-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an alpha-beta parallel-type protein promotes off-pathway species formation.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: Sanne M
authors.initials: SM
authors.lastname: Nabuurs
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D001059:Apoproteins; D005418:Flavodoxin; D019791:Guanidine