Differential effects of disulfide bond formation in TEM-1 versus CTX-M-9 ß-lactamase.

To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two ß-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX-M-9 is two-state whereas TEM-1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9. This modest change to sequence modulates the stabilities-and therefore populations-of TEM-1's equilibrium states and, more surprisingly, creates a novel third state in CTX-M-9. Unlike TEM-1's partially folded intermediate, this third state is a higher-order oligomer with reduced cysteines that retains the native fold and is fully active. Sub-denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX-M-9, in contrast with the observed stabilization in TEM-1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo-oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution.

Tools and tactics to define specificity of metabolic chemical reporters.

Metabolic chemical reporters (MCRs) provide easily accessible means to study glycans in their native environments. However, because monosaccharide precursors are shared by many glycosylation pathways, selective incorporation has been difficult to attain. Here, a strategy for defining the selectivity and enzymatic incorporation of an MCR is presented. Performing ß-elimination to interrogate O-linked sugars and using commercially available glycosidases and glycosyltransferase inhibitors, we probed the specificity of widely used azide (Ac4GalNAz) and alkyne (Ac4GalNAlk and Ac4GlcNAlk) sugar derivatives. Following the outlined strategy, we provide a semiquantitative assessment of the specific and non-specific incorporation of this bioorthogonal sugar (Ac4GalNAz) into numerous N- and O-linked glycosylation pathways. This approach should be generally applicable to other MCRs to define the extent of incorporation into the various glycan species.

Natural and Synthetic Suppressor Mutations Defy Stability-Activity Tradeoffs.

Thermodynamic stability represents one important constraint on protein evolution, but the molecular basis for how mutations that change stability impact fitness remains unclear. Here, we demonstrate that a prevalent global suppressor mutation in TEM ß-lactamase, M182T, increases fitness by reducing proteolysis in vivo. We also show that a synthetic mutation, M182S, can act as a global suppressor and suggest that its absence from natural populations is due to genetic inaccessibility rather than fundamental differences in the protein's stability or activity.