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 (betaalpha)1-8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a approximately 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 (alpha1beta2) and a large cluster (beta5alpha5beta6alpha6beta7) and that these clusters form cores of stability in Ia and Ibp The strongest protection in both states resides in beta4alpha4 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 alpha-helix propensity for beta4, preceded by a highly conserved betaalpha-hairpin clamp that links beta3 and beta4. 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.