Folding of the Pit1 homeodomain near the speed limit

Abstract

Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 μs to form a wellcharacterized intermediate, which rearranges in 17 μs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 μs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10 5 s −1 in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10 5 s −1 at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.activation energy | barrier-limited | chevron plot | dynamics T he homeodomain superfamily is comprised of small threehelical-bundle proteins (1). Their pathways of folding are among the best characterized, with information coming from the effects of point mutations on the folding kinetics of individual members and by comparison of the different members of the family with the same fold but grossly different sequences (2). The mechanism of folding slides from a clear framework mechanism for the engrailed homoeodomain (EnHD) to nucleation-condensation for c-Myb (1, 3). EnHD is an ultrafast folding protein that forms an on-pathway intermediate in 1.7 μs, which rearranges in 17 μs to the native structure. The mechanism is unusually very well-established because the structure of the intermediate has been solved by NMR; it consists of helices 2 and 3 (H2, H3) in native-like conformation with H1 being helical but undocked (4). The H2-turn-H3 motif, is in fact, a stable domain of EnHD (5). Temperature-jump experiments using both IR to monitor secondary structure formation, and Trp fluorescence for tertiary interactions have been used to follow the folding of the wild-type protein, a mutant in which the H1 does not dock at physiological ionic strength, and an H2-turn-H3 construct (5). The H2-turn-H3 domain forms both secondary and tertiary structure in 1.7 μs, and the docking of H1 takes 17 μs. Direct mea...

Malleability of folding intermediates in the homeodomain superfamily

“…The value of ΔG D-N for wild type calculated from the global fit is 4.2 kcal mol −1 , and from the free fit, with a larger error, 4 kcal mol −1 . 2 H∕H-exchange data previously gave a value of 4-5 kcal mol −1 (14).…”

“…The folding kinetics of wild-type Pit1 resembles that of EnHD, which folds via a discrete intermediate in which the HTH motif is an autonomous, stable domain, whose formation and stability are uncoupled from that of H1 (14). But, a detailed Φ-value analysis shows clearly that formation of its intermediate has the formation of all three helices closely coupled.…”

“…We have found that Pit1, the 63-residue homeodomain from pituitary-specific transcription factor (13), folds in better separated phases of 2.3 and 46 μs with more convenient fluorescence changes from its endogenous Trp probe, Trp48, analogous to that in EnHD (14). The 2.3-μs phase is a partly diffusion-limited collapse of the denatured state to give a folding intermediate, I.…”

Human Pluripotent Stem Cells: Applications and Challenges in Neurological Diseases

“…Recent advances in stem cell technology have led to the emergence of methods for differentiation of PSCs into multipotent retinal progenitor cells (RPCs), retinal pigment epithelium (RPE), and photoreceptor-like cells (Buchholz et al, 2009; Meyer et al, 2009, 2011; Osakada et al, 2009; Lamba and Reh, 2011). In addition, disease-specific iPSCs have been derived from patients affected by RP (RP-iPSCs) carrying mutations in RP1 , RP9 , PRPH2 , or RHO genes (Jin et al, 2011; Tucker et al, 2011). Interestingly, in contrast with their normal counterpart, RP-iPSC-derived rod photoreceptor cells degenerated with extended culture period.…”

“…Importantly, the degeneration of rod photoreceptors carrying RP9 mutations was counteracted by the antioxidant α-tocopherol but not in those carrying RP1 , PRPH2 , or RHO mutations supporting the idea that the efficacy of the molecule depends on the genetic mutations (Jin et al, 2011). In an other study, the genetic analysis of the RP-iPSCs lead to the identification of the cilia-related gene male germ cell-associated kinase ( MAK ) gene as a cause of RP (Tucker et al, 2011). Similarly, iPSCs has been established from patients affected by gyrate atrophy, an autosomal recessive eye disease characterized by progressive loss of vision due to retinal degeneration.…”