Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pK_a values

Abstract

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.

Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1)

“…An ensemble of structures of NUPR1, either from wild-type or Thr68Gln mutant, was obtained following a protocol previously reported for IDPs (45,46), with the protein structure initially built in extended conformation and then collapsed in a long-term run (40 ns) performed in the presence of explicit water molecules. Simulation of peptide/NUPR1 complexes were built starting from unbound NUPR1 with equilibrated gyration radius (2.2 nm), as it was experimentally measured (24).…”

The C Terminus of the Ribosomal-Associated Protein LrtA Is an Intrinsically Disordered Oligomer

“…In contrast, the conformations sampled at sufficiently large intervals (e.g., every 0.1 ns) were relatively different in terms of their three-dimensional arrangement, due to the dynamics of C-LrtA. Although not being long enough to obtain a complete statistical ensemble of conformations, the simulation was not prolonged to prevent well-known artifacts, due to over-compaction of the protein [23,24], because a small drift was observed in its size (decrease of R g was ~ 0.3 Å/ns on average, Figure 3).…”

“…A model of C-LrtA (without the His-tag) was obtained in MD simulations by using a protocol previously adopted for IDPs [23,24]. In brief, simulations were performed with the GROMACS package [53] starting from a protein model built by using VMD [25], and collapsing it in a brute-force run carried out in the isobaric-isothermal ensemble.…”

The influence of random-coil chemical shifts on the assessment of structural propensities in folded proteins and IDPs

“…For Asp and Glu residues, this might be a smaller issue as peptide and protein studies are either carried out under very acidic condition (pH < 3), where even the acidic sidechains of these two amino acids are almost completely protonated, or, at neutral pHs, where both are close to completely deprotonated. [122][123][124][125] In contrast, histidine has typical pK a values between 6 and 7 and is therefore partially protonated under close to physiological conditions, making it fall into a "blind range" of binary pH-correction schemes.…”