Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength

Abstract

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein

“…In most protein folding reactions, intermediates are seen to form very rapidly, usually forming too fast to measure. 12,19,24,29,39,69,[73][74][75][76][77][78] Consequently, U and I are effectively at equilibrium before further folding occurs, and it becomes very difficult to establish whether I is on the direct pathway from U to N or whether it is a nonproductive side-product that has to unfold back to U for folding to proceed to N. [26][27][28] In the current study, I is seen to form slowly enough for the rate constants of its formation and disappearance to be precisely determined. The individual rate constants for the formation of N, from I on one pathway and from C on the other pathway (Scheme 1), are about the same (Figures 2 and 3; Table 1) and hence, equal to the net rate constant for the formation of N. The observation that the rate constant of the disappearance of I is the same as the rate constant of the formation of N, is very strongly supportive of I being a productive on-pathway intermediate (Table 1).…”

Proteins: “Boil ’Em, Mash ’Em, Stick ’Em in a Stew”

“…Two-state models also break down when two phase boundaries lie close to one another (top left in Figure B), and multiple states can coexist. Such “third states” can lie on the pathway for folding, − or they can act as traps that hinder refolding because the protein must unfold again before resampling configurations to finally fold. , …”

“…Such "third states" can lie on the pathway for folding, 29−31 or they can act as traps that hinder refolding because the protein must unfold again before resampling configurations to finally fold. 32,33 Protein−protein surface interactions are in many ways analogous to folding: they are still driven by matching of hydrophobic surfaces, even though polar and electrostatic interactions also play a role, 34 and just as folding often produces marginally stable native states to preserve functionally useful fluctuations, protein−protein interactions are not necessarily optimal for function when they are strongest. Strong binding and having many binding partners are mutually exclusive due to the limited information capacity of a protein surface to encode such interactions.…”

The cofactor-dependent folding energy landscape of a light-sensing protein revealed by single-molecule pulling experiments

“…Previous bulk studies have investigated the role of cofactor binding on the folding of flavoproteins that contain the cofactor flavin mononucleotide (FMN). These studies showed that flavoproteins can form molten globules or fold into their native state independently of the cofactor [11][12][13] or fold into an intermediate that is stabilized by FMN binding before reaching the native state 14 . More recently, single molecule manipulation methods have enabled the direct kinetic characterization of intermediates along the folding pathway of small, single-domain proteins that have metal cofactors [15][16][17][18][19] However, these methods have not been applied to proteins that bind to flavin cofactors, let alone multi-domain proteins that bind one of the most common large cofactors known, flavin adenosine diphosphate (FAD).…”