Abstract
The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)1-8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in Ia and Ibp The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.
Highlights
The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces
The motif supports a wide variety of essential enzymatic transformations in all three superkingdoms of life [6,7,8] and is one of the 10 ancestral protein folds that were instrumental in the transition from RNA–protein world to the last universal common ancestor of life (LUCA) to the present complex DNA–RNA–protein world [9, 10]
We have previously examined the relationships between sequence, structure, and fitness in a yeast-based competition assay for three thermophilic indole-3-glycerolphosphate synthase (IGPS) orthologs from the TIM barrel family [17]
Summary
The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Folded states on the landscape often contain elements of the native topology and connect the nascent unfolded polypeptide chain to the functional folded conformation [1, 2] Proteins and their folding pathways have evolved over billions of years, responding to evolutionary forces such as mutation and natural selection [3,4,5]. The pairwise sequence conservation across the family of TIM barrels is typically ∼30%, their folding mechanisms are complex and highly conserved [11] Folding intermediates, both on the productive folding pathway and as misfolded, kinetic traps have been observed for candidate TIM barrels from several bacterial and archaeal organisms [11,12,13,14,15,16]. Significant correlations between the archaeal Sulfolobus solfataricus (SsIGPS) and the bacterial Thermotoga maritima (TmIGPS) and Thermus thermophilus
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