TY - JOUR

T1 - Specific and nonspecific interactions in ultraweak protein−protein associations revealed by solvent paramagnetic relaxation enhancements

AU - Johansson, Helle

AU - Jensen, Malene Ringkjøbing

AU - Gesmar, Henrik

AU - Meier, Sebastian

AU - Vinther, Joachim Møllesøe

AU - Keeler, Camille

AU - Hodsdon, Michael E.

AU - Led, Jens J.

PY - 2014

Y1 - 2014

N2 - 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,non-specific protein–protein interactions often remain elusive. We have investigated the weak selfassociationof human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagneticrelaxation enhancement (PRE) of the amide protons induced by the soluble paramagneticrelaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are inagreement 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 aretaken into account. Secondly, by measuring the PREs of the hGH amide protons at increasing hGHconcentrations and a constant concentration of the relaxation agent, it is shown that a distinctioncan be made between residues that are affected only by transient, non-specific protein–protein interactionsand residues that are involved in specific protein-protein associations. Thus, the PREs ofthe former residues increase linearly with the hGH concentration in the entire concentration rangebecause of a reduction of the diffusion caused by the transient, non-specific protein-protein interactions,while the PREs of the latter residues increase only at the lower hGH concentrations butdecrease at the higher concentrations because of specific protein-protein associations that impedethe access of gadodiamide to the residues of the interaction surface. Finally, it is found that theultra-weak aggregation of hGH involves several interaction sites that are located in patches coveringa large part of the protein surface.

AB - 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,non-specific protein–protein interactions often remain elusive. We have investigated the weak selfassociationof human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagneticrelaxation enhancement (PRE) of the amide protons induced by the soluble paramagneticrelaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are inagreement 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 aretaken into account. Secondly, by measuring the PREs of the hGH amide protons at increasing hGHconcentrations and a constant concentration of the relaxation agent, it is shown that a distinctioncan be made between residues that are affected only by transient, non-specific protein–protein interactionsand residues that are involved in specific protein-protein associations. Thus, the PREs ofthe former residues increase linearly with the hGH concentration in the entire concentration rangebecause of a reduction of the diffusion caused by the transient, non-specific protein-protein interactions,while the PREs of the latter residues increase only at the lower hGH concentrations butdecrease at the higher concentrations because of specific protein-protein associations that impedethe access of gadodiamide to the residues of the interaction surface. Finally, it is found that theultra-weak aggregation of hGH involves several interaction sites that are located in patches coveringa large part of the protein surface.

KW - Faculty of Science

U2 - 10.1021/ja503546j

DO - 10.1021/ja503546j

M3 - Journal article

C2 - 24969589

VL - 136

SP - 10277

EP - 10286

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 29

ER -