Facile detection of protein-protein interactions by one-dimensional NMR spectroscopy.
Two methods for detecting protein-protein interactions in solution using one-dimensional (1D) NMR spectroscopy are described. Both methods rely on measurement of the intensity of the strongest methyl resonance (SMR), which for most proteins is observed at 0.8-0.9 ppm. The severe resonance overlap in this region facilitates detection of the SMR at low micromolar and even sub-micromolar protein concentrations. A decreased SMR intensity in the 1H NMR spectrum of a protein mixture compared to the added SMR intensities of the isolated proteins reports that the proteins interact (SMR method). Decreased SMR intensities in 1D 13C-edited 1H NMR spectra of 13C-labeled proteins upon addition of unlabeled proteins or macromolecules also demonstrate binding (SMRC method). Analysis of the interaction between XIAP and Smac, two proteins involved in apoptosis, illustrates both methods. A study showing that phospholipids compete with the neuronal core complex for Ca2+-dependent binding to the presynaptic Ca2+-sensor synaptotagmin 1 illustrates the usefulness of the SMRC method in studying multicomponent systems.
Araç
Demet
D
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA.
Murphy
Tara
T
Rizo
Josep
J
D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012996:Solutions; D050863:Synaptotagmin I; D050857:Synaptotagmins; D051636:X-Linked Inhibitor of Apoptosis Protein
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D012996:Solutions; D050863:Synaptotagmin I; D051636:X-Linked Inhibitor of Apoptosis Protein; D050857:Synaptotagmins