Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

Significance Rigidifying flexible sites is a powerful method to improve enzyme stability. However, if the highly flexible regions form the active site, modifying them risks losing activity due to the activity–stability trade-off. We hypothesized here that regions outside the active site whose dynamics were highly correlated to flexible active sites, would provide good targets for stabilizing mutations. To test this hypothesis, six variants were constructed in the 3M variant of Escherichia coli transketolase. The best variant had a 10.8-fold improved half-life at 55 °C, and increased the Tm and Tagg by 3 °C and 4.3 °C, respectively. The variants even increased the activity, by up to threefold. This study highlights how protein engineering strategies could be potentially improved by considering long-range dynamics.

Interactions network predictions. The RING 2.0 web server was applied for predicting residue interaction networks (1). Wild-type (1QGD.pdb) and 3M variant (5HHT.pdb) structures were used as the input. The distance thresholds were set as the option of Relaxed, which corresponds to the distance thresholds including hydrogen bond (5.5 Å), salt bridge (5.0 Å) disulphide bond (3.0 Å), Van-der-Waals (0.8 Å) and π-π stacking (7.0 Å). During the calculation, the network policy was set as the Closest and the interactions with water and hetero atoms were not considered. Only the most probable interaction type was calculated between a residue pair. The output files were imported to Cytoscape for further analysis (2) .
Thermal transition mid-point (Tm) and aggregation onset (Tagg) temperatures. Intrinsic protein fluorescence (IPF) (266 nm excitation, 280-450 nm emission scan) and static light scattering (SLS) at 266 nm and 473 nm, were measured simultaneously for measuring the Tm and Tagg values of TK variants using a UNit (Unchained Laboratories, Pleasanton CA). Measures were taken as a function of temperature in the range of 30-90 o C with steps of 1 o C, equilibration time of 30 s at each temperature. The microcuvette arrays were loaded with 9 µL of 0.1 mg/mL sample and all experiments were performed in triplicates. Tagg values were determined with instrument software by the analysis of SLS counts at 266 nm. Fluorescence intensity ratio determined between the spectral intensity at 350 nm to that at 330 nm was used to calculate melting temperature, Tm. The fluorescence intensity ratio was fitted to a two-state transition model using equation 2 (3,4), in the software OriginPro 9.0 (Origin Lab Corp., Northampton, MA, USA).
where IT is the observed signal, IN and ID are the native and denatured baseline intercepts, a and b are the native and denatured baseline slopes, T is the temperature, ∆Hvh is the van't Hoff enthalpy, R is the gas constant (1.987 cal mol -1 K -1 ) and Tm is the apparent mid-point of the observed thermal transition. The van't Hoff entropy is calculated using equation 3.
The mole-fraction, fT, of unfolded protein at any temperature T, was calculated from = − − and by substituting for IT in the equation 2, this reduces to Eq. 5

Results
Why did 5M/I365L have the highest kinetic stability and Tagg, but modestly increased Tm? It is not easy to explain why 5M/I365L had the longest half-life at 55 o C, whereas its Tm was not the highest among the variants (Table 2). Some differences between 5M/I365L and the other variants were observed, and these may have contributed in some way to its outlier behaviour. First, 5M/I365L decreased the flexibility at G331-K347, which was not found for other variants of 3M (Fig. 4 C & S7). The G331-K347 fragment was in close proximity to the mutation I365L (Fig. S8). It was also one of the most flexible regions in the TK Pyr-domain (323-539 aa). The flexibility of the whole Pyr-domain for 5M/I365L was lower on average than for other variants, which may have been critically linked to stabilization at G331-K347 (Fig. S7). On the other hand, in the PP-domain (2-322 aa) of the 5M/I365L, several regions around residues 94, 192, 254 and 282 became more flexible than in 3M and other variants (Fig. S7). Unlike other variants, 5M/I365L had a relatively more stable Pyr-domain and a less stable PP-domain compared to other variants including 7M and 5M/G506A.   S2. Residue interaction networks of the local region around the 3M mutation sites for WT and 3M. A, Interaction network for WT calculated using 1QGD.pdb. B, Interaction network for 3M calculated using 5HHT.pdb. Nodes corresponding to residues are coloured by red (mutation sites), green (residues directly connecting with at least one mutation site), grey (residues indirectly connecting with mutation sites). Edges corresponding to non-covalent interactions are coloured as solid black lines (Van der Waals), blue dashed lines (hydrogen bonds), red dashed lines (salt bridges), pink dashed lines (π-π stacking).