Specific and Nonspecific Interactions in Ultraweak Protein–Protein Associations Revealed by Solvent Paramagnetic Relaxation Enhancements

Abstract

Weak and transient protein–protein interactions underlie numerous biological processes. However, the location of the interaction sites of the specific complexes and the effect of transient, nonspecific protein–protein interactions often remain elusive. We have investigated the weak self-association of human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagnetic relaxation enhancement (PRE) of the amide protons induced by the soluble paramagnetic relaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are in agreement with the general Hwang-Freed model for relaxation by translational diffusion (J. Chem. Phys.1975, 63, 4017–4025), only if crowding effects on the diffusion in the protein solution are taken into account. Second, by measuring the PREs of the amide protons at increasing hGH concentrations and a constant concentration of the relaxation agent, it is shown that a distinction can be made between residues that are affected only by transient, nonspecific protein–protein interactions and residues that are involved in specific protein–protein associations. Thus, the PREs of the former residues increase linearly with the hGH concentration in the entire concentration range because of a reduction of the diffusion caused by the transient, nonspecific protein–protein interactions, while the PREs of the latter residues increase only at the lower hGH concentrations but decrease at the higher concentrations because of specific protein–protein associations that impede the access of gadodiamide to the residues of the interaction surface. Finally, it is found that the ultraweak aggregation of hGH involves several interaction sites that are located in patches covering a large part of the protein surface.

Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell‐mimicking crowded solution

“…In particular, Johansson et al . , were able to identify residues of human growth hormone that are involved in either unspecific or specific protein self‐interactions based on the PRE profile of amide protons induced by gadodiamide (a gadolinium‐based paramagnetic relaxation agent, also referred to as Gd‐DTPA‐BMA). Gadodiamide‐induced PRE effects on Ub were previously interpreted according to a relaxation model where the relaxation agent forms an unspecific, yet rotationally correlated, complex with the protein .…”

Computational study of the inhibitory mechanism of the kinase CDK5 hyperactivity by peptide p5 and derivation of a pharmacophore

“…The method is thus designed to account for conformational selection of both molecules and for mutually induced fit upon association. The method is also designed to identify weak and ultra-weak modes of association, which have been shown to play a role in molecular recognition [26, 27]. The method yields a set of conformational families of the complex, from which a discrete set of conformations (the “binding modes”) are identified.…”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…79,130,131 It is not clear, however, whether such soft interactions simply hinder diffusion and alter the conformational energy landscape as discussed above or whether interactions between biomolecules are altered with possible functional implications. 16,66,70,74,84,132 Crowding has been found to affect native complex formation 31,68,133−135 and aggregation, 136−138 while other evidence indicates enhanced formation of nonspecific complexes between proteins in the cellular milieu.…”