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Amphiphilic nanoparticles are highly biocompatible frameworks widely used for shuttling hydrophobic molecules across hydrophilic cellular environments. Their use can also span applications focused on achieving a selective binding against a target receptor. However, the molecular factors leading to an optimal binding often remain a critical step to assess. Here we have evaluated through advanced sampling free-energy simulations, combined with electronic circular dichroism spectra predictions, the binding of an amphiphilic polyethylene glycol (PEG)-based nanoparticle incorporating folate monomers with human folate receptor alpha. It turns out from the free-energy predictions that the optimal binding is achieved when the receptor target and the ligand-mimicking nanoparticle have a comparable size and the shortest spacer length of the folate monomer. Furthermore, we calculated the ECD spectrum of the folate monomer binding the target receptor, as well as that of the binding pocket of the receptor. Noteworthy, only when the optimal binding is achieved, the two ECD spectra closely match each other. Shape factors control the binding efficacy of nanoparticles with target receptors.

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 pKa 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 pKa 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.

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr, and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr, HPr, comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr. This implies that specific fragments of Rsd and HPr can be used to interfere with other protein-protein interactions (PPIs) of each other protein.