NMR techniques for identifying the interface of a larger protein-protein complex: cross-saturation and transferred cross-saturation experiments.
NMR provides detailed structural information for protein complexes with molecular weights up to 30 kDa. However, it is difficult to obtain such information on larger proteins using NMR. To identify the interface of a complex with a molecular weight of over 50 kDa, chemical shift perturbation or hydrogen-deuterium (H-D) exchange experiments have been frequently used. The binding sites determined by these methods are quite similar, but not identical, to the contact surface identified by X-ray crystallography. The difference in the binding sites can be explained by the fact that the chemical shift and H-D exchange rates are affected by various factors, such as changes in the microenvironment and subtle conformational changes induced by the binding. Therefore, an alternative NMR strategy is required to identify the interaction site in large protein-protein complexes. The cross-saturation experiment is an NMR measurement for precise identification of the interface of larger protein complexes. This method extensively utilizes deuteration for proteins and the cross-saturation phenomenon along with TROSY detection. In this chapter, the principle of the cross-saturation experiment will be illustrated and then the extended version of the method, transferred cross-saturation, and its applications to larger protein complexes will be demonstrated.
Shimada
Ichio
I
Division of Physical Chemistry, Graduate School of Pharmaceutical Science, The Universityof Tokyo, Hongo, Japan.
D000818:Animals; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013211:Staphylococcus aureus / Q000276:immunology / Q000378:metabolism; D014867:Water / Q000737:chemistry
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D011506:Proteins; D013205:Staphylococcal Protein A; D014867:Water; D003094:Collagen