Fig 1.
Overview of the modeled BglB-pNPG complex showing positions mutated in this study and reaction used to determine functional properties of individual mutants.
PyMOL rendering [32] of modeled BglB in complex with pNPG showing the 68 sequence positions selected for mutation in this study (teal spheres) and the modeled transition-state structure (white ball and stick model). Below, reaction scheme of the hydrolysis of pNPG by BglB used to determine functional Tm and kinetic parameters kcat, KM, and kcat/KM.
Fig 2.
Relative effects on enzyme functional parameters for 129 mutants of BglB.
Each mutant gets a bar with six colored boxes, depicting 1) soluble protein expression, 2) Tm, 3) kcat, 4) KM, 5) kcat/KM, and 6) conservation within Pfam GH01. For expression (box 1), a black box indicates soluble expression > 0.10 mg/mL, and a white box indicates expression < 0.10 mg/mL in E. coli BLR (DE3). For Tm (box 2), a linear scale is used to depict change in Tm compared to wild type, with mutants with greater Tm in green, and those with lower Tm in purple. For kcat, 1/KM, and kcat/KM (boxes 3–5), blue indicates lower values, and orange indicates higher values relative to the wild type value, as indicated by the color legend (top). For conservation (box 6), the frequency of native BglB residue in an alignment of the BglB sequence to 1,554 sequences from Pfam GH01 is shown, on a linear scale from 0% to 100%. The quantitative measurements used to produce this illustration are provided in S1 Table.
Fig 3.
Structural analysis of Rosetta models of designed point mutants of BglB with effects on thermal stability.
Four mutant panels are shown, sorted from left to right by increasing Tm. In the top panel, experimentally-determined change in Tm and kcat/KM are given. For reference, the Tm for the wild type sequence is 39.9°C, and the kcat/KM is 174,000 M–1min–1. In the next panel down, sequence logos depict the local area of sequence conservation based on an alignment of 1,544 sequences from Pfam GH01. At bottom, depictions of the local area of the mutation in the BglB WT protein (top) and RosettaDesign model of mutation (bottom).
Fig 4.
Relationship between protein melting temperature (Tm) and kinetic constants kcat, KM, and kcat/KM in the BglB system.
Tm values are on a linear scale in units of degrees Celsius and values for kinetic constants are on a log scale, with units of min–1, mM, and M–1min–1, respectively. These parameters are not correlated in BglB (Pearson correlation < 0.25 for Tm versus each of the kinetic constants kcat, KM, and kcat/KM). The independence of these parameters suggests that they can be separately engineered in a rational manner.
Fig 5.
Correlations between conservation within functional protein family and enzyme functional parameters protein melting temperature (Tm) and kinetic constants (kcat, KM, and kcat/KM) in the BglB system.
Scatter plots showing conservation analysis from an alignment of 1,554 proteins in Pfam family 1 (glycoside hydrolases) versus measured values for Tm (linear scale, units of °C) and each of the kinetic constants kcat, KM, and kcat/KM (log scale) with units of min–1, mM, and M–1min–1, respectively.
Fig 6.
Correlations between experimentally-determined Tm and structural features from molecular modeling algorithms.
For each of the three computational protocols used for prediction of stability in this study, the two most-correlated (black) and two least-correlated (gray) features are plotted against experimentally-determined Tm. Pearson correlation between the two sets of values is provided above each plot. For descriptions of individual features for each of the three algorithms, see references for RosettaDesign [11], Rosetta ΔΔG [12], and FoldX [9].