Design, Synthesis And Characterization Of Paramagnetic Tags In Site-specific Labeling Of Proteins

X-ray crystallography and nuclear magnetic resonance(NMR) are the mostly used techniques in obtaining the three-dimensional structure of biological macromolecules with atom resolution, Compared with X-ray crystallography, NMR has advantages in:(1) the proteins is not ncecessarily crystallized;(2) the measurement condition is close to physiological environment;(3) NMR is able to investigate the dynamic properties of biomolecues in solution. NOEs and dihedral angles are the main structural constraints in structural biology by virtue of NMR. intermolecular NOEs are difficult to assign in many protein-protein and tranditional NMR becomes awkard in study of domain displayment in multiple-domain proteins.Site-specific labeling of proteins with paramagnetic ions especially lanthanides greatly enhances the power of NMR in structural biology. The dipolar interactions between unpaired electrons and nuclei spins of protein provide a rich source of structural information of proteins. At present, chemically synthesized tags are almost invariably attached to the protein by formation of one or two disulfide bonds. In this study, we report a new method of site-specific tagging proteins with paramagnetic molecules vis Michael addition reaction. We first designed and synthesized a series of new paramagnetic tags which contain difunctional groups, and investigated the kinetics of the reaction between paramagnetic tags(Ll, L2) and L-Cysteine. We show that L1and L2are ideals paramagnetic tag in labeling of proteins. And significant pseudocontact shift(PCS) were observed in the L1/L2derivatized protein adducts. In summary, we show that thiol-ene like Michael addition reaction is a valuable approach in site-specific labeling of proteins with chemically inert paramagnetic tags.