Figure 4. Experimental assessment of the role of the intermediate state determined in this work in the product release process.

(A) Structure of the unlocked state illustrating the hydrogen bond between side chains of N44 and E35 that stabilises this intermediate species. (B) The hydrogen bond is not formed in the locked state because N44 and E35 are too far apart. (C) The N44A variant, which lacks the hydrogen bond donor, is unable to form this hydrogen bond, thus destabilising the intermediate state and inhibiting the release of the product. The decrease of the ability of the N44A mutant to release triNAG has been assessed by surface plasmon resonance (SPR) experiments. (D) Cellular assay of lysozyme activity. The N44A variant has an intermediate activity between wild type and the control E35D variant.

DOI: http://dx.doi.org/10.7554/eLife.02777.011

Figure 4—figure supplement 1. Comparison of the ¹H-¹⁵N HSQC spectra of WT (black) and N44A mutant (red).

DOI: http://dx.doi.org/10.7554/eLife.02777.012

Figure 4—figure supplement 2. Comparison of the free-energy landscapes of wild-type (red) and N44A mutant (black) lysozyme.

In the N44A mutant, the unlocked state (see also Figure 1) is absent. Free-energy landscapes were obtained as described in Figure 1.

DOI: http://dx.doi.org/10.7554/eLife.02777.013

Figure 4—figure supplement 3. Study of the N46Q/V110Q mutant.

(A) Illustration of the engineered N46Q/V110Q glutamine–glutamine interactions. (B) The ¹H-¹⁵N-HSQC spectrum of the N46Q/V110Q mutant shows that the mutation does not affect the structural properties of the mutant (see also Figure 4—figure supplement 1). (C) The increase in the ability of the N46Q/V110Q mutant to release triNAG has been assessed by surface plasmon resonance (SPR) experiments.

DOI: http://dx.doi.org/10.7554/eLife.02777.014