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Abstract

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

An allosteric site in the T-cell receptor Cβ domain plays a critical signalling role

“…To characterize the α/β-interface of the B4.2.3 TCR in solution, we utilized a TROSY-based cross-saturation transfer experiment34 in which TCR was prepared with 15 N, 2 H-labelled β-chain and unlabelled (protonated) α-chain. Here selective irradiation of the aliphatic protons of the α-chain is expected to transfer to β-chain amides located at the interface with the α-chain through cross-relaxation.…”

“…To determine the surface of the β-chain affected by the α-chain in solution, 2D cross-saturation transfer experiments34 were performed on a 180 μM B4.2.3 TCR sample prepared with U-[ 15 N, 2 H]-labelled β-chain and unlabelled (protonated) α-chain in 50 mM NaCl, 25 mM HEPES pH 7.3, 0.01% NaN 3 , 1 U Roche protease inhibitor in 90% H 2 O/10% D 2 O. Acquisition parameters for the 2D 1 H- 15 N TROSY-HSQC were 256 and 2,048 complex points in the 15 N, 1 H N dimensions with acquisition times of 45 ms, 71 ms and 16 scans recorded at 800 MHz, 25 °C.…”

A Receptor-based Switch that Regulates Anthrax Toxin Pore Formation

“…In order to limit spin diffusion in the 15 N-labeled protein, it was extensively deuterated by growing the cells in 100% D 2 O minimal media using 2 H-glucose as the sole carbon source [32]. The labeled VWA-domain was purified to homogeneity as described under Materials and Methods and was unfolded to protonate the residues within the protein core and refolded to increase the number of cross-peaks in the [ 15 N, 1 H] TROSY-HSQC spectrum.…”

“…The aliphatic proton resonances of the unlabeled protein are then saturated with a brief radiofrequency pulse and this saturation is transferred selectively to contact residues of the 2 H, 15 N-labeled protein by spin diffusion. Consequently, the intensity of amide cross-peaks representing labeled residues that lie at the protein-protein interaction surface are selectively reduced by cross-relaxation [32], [33]. Here we have used this technique to obtain evidence for a new toxin-receptor intermediate in the pathway leading to pore formation and show that the receptor remains attached to PA domain 4 following low pH-dependent conversion.…”

Nuclear magnetic resonance analysis of protein–DNA interactions

“…Cross-saturation experiments were first introduced to identify the interfaces of large protein -protein complexes [28] and have been successfully applied to mapping out the binding surfaces in protein -nucleic acid complexes [29,30]. This method is based on selective saturation of protons from one molecule partner that leads to a decrease in magnetization throughout the molecule, which is mostly controlled by spin diffusion.…”