Visualization of transient ultra-weak protein self-association in solution using paramagnetic relaxation enhancement.
Ultra-weak macromolecular self-association is exceptionally difficult to both detect and visualize using conventional biophysical techniques owing to the very low population of the associated species, yet such weak intermolecular interactions coupled with nucleation events play an important role in driving spontaneous self-assembly to form higher-order architectures (such as crystals, viral capsids, and amyloid fibrils). In this article, we detect and characterize transient, ultra-weak self-association (KD >or= 15 mM) involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), using EDTA-Mn2+ conjugated at three separate sites (E5C, E25C, and E32C, one at a time). Large intermolecular PRE effects, above the background observed with hydroxylamine-EDTA-Mn2+ as a control, are observed for two of the three paramagnetically labeled sites, E5C and E32C. The extent of self-association can be modulated (significantly reduced) by increasing the ionic strength or by the introduction of a negative charge (S46D mutation) within a positively charged surface patch, and abolished upon the addition of the N-terminal domain of enzyme I (EIN) to form a specific EIN-HPr complex. The PRE profiles observed for E5C and E32C can be fitted simultaneously and accounted for quantitatively using conjoined rigid body/torsion angle dynamics-simulated annealing with an ensemble of states to represent the distribution of one molecule of HPr relative to its partner.
Tang
Chun
C
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Ghirlando
Rodolfo
R
Clore
G Marius
GM
D001426:Bacterial Proteins / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D019811:Hydroxylamine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008280:Magnetics; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D009994:Osmolar Concentration; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011485:Protein Binding; D012996:Solutions / Q000737:chemistry; D013499:Surface Properties
D001426:Bacterial Proteins; D012996:Solutions; D019811:Hydroxylamine; D008345:Manganese; D004492:Edetic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr