Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin-antitoxin partners in order to probe possible cross-talk between these systems.

Association of disease duration and sensorimotor function in type 2 diabetes mellitus: beyond diabetic peripheral neuropathy.

PURPOSE: The aim of the study is to assess association of the duration of diabetes and vibration, proprioception, muscle strength, reaction time and balance measures in people with type 2 diabetes mellitus (DM) without peripheral neuropathy. METHODS: Forty-seven type 2 diabetics without peripheral neuropathy and 23 healthy controls were recruited for the study. Patients with type 2 DM were further classified into 23 patients who suffered from diabetes for less than 5 years (<5yrDM) and 24 patients who had diabetes for 5 years and above (≥5yrDM). All participants were assessed for Michigan neuropathy screening instrument (MNSI), vibration perception threshold (VPT), proprioception, muscles strength, centre of pressure (COP) range, COP sway and reaction time. RESULTS: ≥5yrDM patients were found to significantly differ from healthy control in MNSI score (p ≤ 0.013), VPT score (p ≤ 0.002), reaction time (p ≤ 0.018), COP range (p ≤ 0.005) and COP sway (p ≤ 0.027). A significant difference was found only in reaction time (p < 0.002) except in the back direction (p = 0.089), and COP range (p ≤ 0.016) except in the front (p = 0.101) and right direction (p = 0.085) between <5yrDM patients and healthy controls. CONCLUSIONS: ≥5yrDM patients exhibit a subtle deterioration in VPT, reaction time, and balance measure while <5yr DM patients were impaired only in COP range and reaction time when compared with healthy control.