The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain

Abstract

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th alpha-helical repeat from chicken brain alpha-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th alpha-helical repeat from chicken brain alpha-spectrin). An extensive Phi-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.

Fig. 1

The structure and folding pathway of spectrin R1516. (a) R1516 is a two-domain fragment from chicken brain α-spectrin (from Protein Data Bank file 1U4Q). The chain is shown from blue (A-helix of R15) to red (C-helix of R16). The C-helix of R15 forms a continuous helix with the A-helix of R16. (b) Folding pathway of R1516 adapted from Ref.. The R15 domain folds rapidly to form a stable intermediate with R15 folded and R16 unfolded. The R16 domain folds more slowly than R15. In the unfolding direction, R16 unfolds first. Loss of interactions with R16 destabilises the R15 domain such that this unfolds rapidly.

Fig. 2

Chevron plots of wild type (WT) R1516 and a mutant. The proteins were all purified as previously described. The kinetics were analysed as previously described. (a) For WT R1516, there are three observable folding phases and one observable unfolding phase. The fastest folding phase has approximately 30% of the total amplitude, the intermediate phase has approximately 60%, and the slowest of the three phases (not shown here) has less than 10%. The fast refolding phase has been attributed to the folding of R15 (red closed circles); the middle refolding phase, to the folding of R16 (blue closed circles); and the slowest of the phases, to prolyl isomerisation in WT R1516. The single observable unfolding phase is the unfolding of R16. R15 can be seen to unfold in double-jump experiments. Data were taken from Ref. . (b) Chevron plot for the mutant L97A in the R16 domain of R1516 compared with WT R1516. The unfolding of the R15 domain is unaffected by any of the mutations (shown in red: closed circles, mutant; open circles, WT). The L97A mutation slows the unfolding of the R16 domain slightly but speeds the unfolding of the R16 domain significantly (i.e., the Φ-value is low, 0.3) (shown in blue: closed circles, mutant; open circles, WT). In this case, the R16 domain unfolds so rapidly that both unfolding phases can be observed in a single-jump experiment. In Supplementary Data, chevron plots for the R16 domain in R1516 are shown for all mutant proteins, compared with R16 in WT R1516.

Fig. 3

Comparison of the Φ-values for R16 alone and in R1516. The patterns of the Φ-values are essentially the same whether the R16 domain is in isolation or attached to folded R15. The darker bars are the Φ-values for surface Ala → Gly mutants, which probe secondary structure formation. The lighter bars are for mutations of buried residues to Ala, which probe the tertiary structure. In the early rate-determining transition state (top panel), the magnitude and the patterns of Φ-values are the same. In the late transition state (lower panel), which is only rate determining at high denaturant concentrations, the Φ-values are slightly higher for the R16 domain in R1516, but, again, the patterns of Φ-values are the same. Note that the error in Φ_late is relatively high, especially for R16 in R1516, due to uncertainties in fitting the data where there is little curvature in the unfolding arm (see Refs. and the text). This is likely why there are some nonclassical Φ-values (> 1). Data for R16 alone were taken from Ref. .