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. 2008 Dec;2(6):365-77.
doi: 10.2976/1.2991513. Epub 2008 Oct 15.

Studying the folding of multidomain proteins

Sarah Batey  1 , Adrian A Nickson, Jane Clarke
Affiliations

Studying the folding of multidomain proteins

Sarah Batey et al. HFSP J. 2008 Dec.

Abstract

There have been relatively few detailed comprehensive studies of the folding of protein domains (or modules) in the context of their natural covalently linked neighbors. This is despite the fact that a significant proportion of the proteome consists of multidomain proteins. In this review we highlight some key experimental investigations of the folding of multidomain proteins to draw attention to the difficulties that can arise in analyzing such systems. The evidence suggests that interdomain interactions can significantly affect stability, folding, and unfolding rates. However, preliminary studies suggest that folding pathways are unaffected-to this extent domains can be truly considered to be independent folding units. Nonetheless, it is clear that interactions between domains cannot be ignored, in particular when considering the effects of mutations.

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Figures

Figure 1
Figure 1. Multidomain proteins discussed in this review.
The domains are colored as follows: N-terminal domain, blue; C-terminal domain, red; Middle domain [(e) and (f)], green. In (a) and (d) the linker region between the N-and C-domains is colored green. (a) Crystal structure of fibronectin type III domains FNfn9 and FNfn10 from human fibronectin (pdb code 1fnf). The two-residue linker that is the end of FNfn9 and the start of FNfn10 is shown in green. This linker is important for the stability of both domains. The domains themselves do not interact detectably. (b) The structure of γB-crystallin (pdb code 1amm). There is a large interface between the two domains which stabilizes the folded domains. (c) Semiliki forest virus protein (SFVP, pdb code 1vcq). The two domains fold sequentially. (d) bsPGK (pdb code 1php). The linking helix (green) was not included in either single domain construct. The data suggest that this linking helix is important for the stability of the C-domain. (e) Crystal structure of the 15th, 16th, and 17th spectrin repeat domains from chicken brain α-spectrin (pdb code 1u4q). The C-helix of R15 forms a continuous helix with the A-helix of R16, while the C-helix of R16 forms a continuous helix with the A-helix of R17. (f) The three domain protein trigger factor (pdb code 1w26). An extension of the N-terminal domain forms important contacts with the C-terminal domain.
Figure 2
Figure 2. Four possible equilibrium denaturant profiles for multidomain proteins.
Unfolding transitions of the individual domains are shown in red and blue and the multidomain protein is shown in black. (a) Independent domains with significantly separate [d]50%. Two separate unfolding transitions can be seen in the multidomain protein. (b) Independent domains with similar [d]50%. Only one transition can be observed in the multidomain protein; however, the m-value (the slope) is significantly lower than that of the individual domains. In this case the individual domain transitions can only be determined if the spectroscopic properties are different. (c) Interacting domains with no significantly populated intermediate. There is one observable unfolding transition with an m-value approximately that of the sum of the two individual m-values (mTOT≈mA+mB). (d) Interacting domains with a populated unfolding intermediate. Only one transition is observed; however, the m-value is significantly lower than the sum of the two individual m-values. (a) and (b) use modeled data, (c) and (d) use actual data from spectrin R1617 (Batey et al., 2005) and R1516 (Batey and Clarke, 2006), respectively.
Figure 3
Figure 3. The equilibrium properties of the spectrin tandem repeat domains R1516 and R1617 can be explained using kinetic data.
(a) Equilibrium populations of the folded (red), intermediate (black), and unfolded (blue) species of R1516 determined from the kinetic rate constants. (b) Equilibrium populations of the folded (red), intermediate (black), and unfolded (blue) species of R1617 determined from the kinetic rate constants. (c) Modeled (open circles) and actual (closed circles) equilibrium denaturation curves of R1516 from CD (black) and fluorescence (red) measurements. (d) Modeled (open circles) and actual (closed circles) equilibrium denaturation curves of R1617 from CD (black) and fluorescence (red) measurements. Data taken from (Batey and Clarke, 2006).
Figure 4
Figure 4. Kinetics of the three-domain protein spectrin R151617, in this case domains are affected only by direct neighbors.
The kinetics of R15 (purple), R16 (red), and R17 (blue) shown as individual domains (closed circles) in R1516 (open circles), in R1617 (open triangles), and in R151617 (closed triangles). Top row shows the kinetics of R15 (alone and in the two- and three-domain proteins, R1516 and R151617, respectively). The unfolding of R15 is slowed by its neighbor, R16, in R1516. There is no additional effect on either folding or unfolding rates by the addition of R17 to form R151617. Middle row shows the kinetics of R16 (alone, in the two-domain proteins R1516 and R1617, and in the three-domain protein, R151617). R16 is stabilized in R1516 by an increase in folding rate and a decrease in unfolding rate. In R1617, the folding rate of R16 is slowed due to the population of a collapsed intermediate; the unfolding rate is also decreased. In R151617 the folding rate of R16 is changed by two opposing effects, the presence of folding R15 increases the folding rate, however, the presence of R17 still leads to the population of the collapsed intermediate. The unfolding of R16 in R151617 is slower than in R1516 or R1617 due to the additive effect of the two neighboring domains. The bottom row shows the kinetics of R17 (alone, in the two-domain protein R1617 and in the three-domain protein R151617). R17 is stabilized in R1617 by an increase in folding rate and a decrease in unfolding rate. The kinetics of R17 in R151617 are exactly the same as those in R1617 therefore the addition of R15 has no effect on R17.
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