Table 1.
Characteristics of the enzyme families selected for cluster analysis, with the most thermostable member highlighted in bold.
Fig 1.
Identifying equivalent clusters in homologous proteins allows for direct comparison of local environments.
(A) A cartoon depiction of cluster of adjacent residues is shown (red circle). (B) Structural alignment of paired enzymes is shown, with PDB 1vbr in orange and 2uwf in gray. The structurally aligned residues for the paired enzymes are shown beneath. (C) Differences in atomic packing is depicted with alternate sequences shown in stick and sphere representation on PDB 2wva.
Fig 2.
Evaluating the potential for epistasis.
(A) The number of residues in each motif are determined for all representative thermophilic-mesophilic structure pairs and binned according to the motif size. (B) The number of residue substitutions, given as Hamming distance, in each equivalent thermophilic-mesophilic motif is determined and binned.
Fig 3.
Thermophilic enzyme clusters display closer atomic packing compared to mesophilic enzyme clusters for most enzyme pairs evaluated.
(A) SASA1.4 values for clusters from the representative thermophilic-mesophilic structure pairs are shown, with thermophilic clusters shown in red, mesophilic clusters in green and the difference, ΔSASA1.4, in blue. Values are sorted by ΔSASA1.4. (B) SASA1.4 values are shown comparing clusters from the thermophilic (PDB 1a5z) and mesophilic (PDB 6ldh) lactate dehydrogenase enzymes, which have a difference in optimum activity temperature of 30°C. (C) the thermophilic (PDB 1a5z) and mesophilic (PDB 5ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 48°C, (D) and the thermophilic (PDB 1a5z) and psychrophilic (PDB 1ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 70°C.
Table 2.
Comparing void volumes, as determined by ΔSASA1.4 and residue contact number, and percent sequence identity for paired clusters.
Fig 4.
Anchor residues are conserved in atomic packing.
(A) The thermostable GH9 (PDB 4dod) is shown in surface representation, with anchor residues that are seen in a larger number of clusters shown in stick representation. Residues exhibiting the largest ΔSASA1.4, which are never anchor residues, are colored red. (B) Sequence positions from 4dod are binned by the number of clusters in which they are found. The heat scale indicates ΔSASA1.4. Importantly, blue is not observed as there are no mesophilic clusters with significantly better atomic packing relative to the matched thermophilic cluster. (C) Sequence positions from the representative set of structures are binned by the number of motifs in which they are found (x-axis), with ΔSASA1.4 shown for each paired position (y-axis). A white symbol indicates sequence conservation, and gray indicates the sequence differs at that position.
Fig 5.
The backbone can move significantly in the structurally equivalent clusters.
(A) Three Cα atoms from a paired cluster are shown in red spheres (thermophilic enzyme) and purple spheres (mesophilic enzyme). The atoms are labeled a, b and c for the thermophilic enzyme and a’, b’ and c’ for the mesophilic enzyme. The Euclidian distances between Cα atoms are shown for each enzyme, with the distance differences at right. (B) The sum of the absolute values for the distance differences (red), and the average distance differences (blue) for each representative cluster are shown, sorted by summed or averaged distances.