Solid-state NMR Structural Studies of Proteins Using Cyclen Based Paramagnetic Metal Chelating Probes PDF Download

Author: Rajith Madushanka Jayasinha Arachchige
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Languages : en
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Book Description
The capability of solid-state NMR spectroscopy to provide site-specific atomic-level information makes it one of the main tools used in the structural and dynamic analysis of complex non-crystalline biological macromolecules. However, the challenges associated with measurements of unambiguous long-range distance restraints between nuclear spins by conventional solid-state NMR methods have a significant impact on the ability to determine three-dimensional structures of proteins and nucleic acids. Recently, it has been shown that paramagnetism-based solid-state NMR methods can be utilized to overcome this scarcity of long-range structural information. Electron-nucleus through space dipolar interactions in proteins containing paramagnetic metal centers lead to NMR spectral effects including paramagnetic relaxation enhancements (PREs) and pseudocontact shifts (PCSs), which can provide structural restraints up to 30 A° and beyond exceeding by four to five-fold the length scales accessible by conventional methods. For natively diamagnetic proteins with no endogenous paramagnetic metal centers can be introduced by covalently linking to the protein small molecule tags containing high affinity metal binding motifs. The focus of this research was to develop new thiol-reactive mono and bidentate compact paramagnetic metal chelating probes based on the cyclen manifold for improved solid-state NMR PRE and PCS measurements in natively diamagnetic proteins. The single arm TETAC tag was able to overcome the key shortcomings associated with the more bulky and flexible EDTA type metal-binding tags used in initial paramagnetic solid-state NMR studies. Relative to PREs, the magnitude of which depends primarily on the electron-nucleus distance, the measurements of PCS effects are far more challenging due to an additional dependence on the location of the nucleus in the frame of the g-tensor of the unpaired electron spin of the paramagnetic center. Specifically, such measurements are susceptible to the flexibility of the paramagnetic tag, which can considerably reduce the PCS magnitude or average the effect altogether. To alleviate this problem compact cyclen based tags, which can rigidly be attached to the protein via two disulfide bridges, were designed. The new double arm tags, 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)bis(N-(2-(pyridin-2-yldisulfanyl)ethyl)acetamide) and 2,2'-(4,10-bis(2-(pyridin-2-yldisulfanyl)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid, are able to immobilize paramagnetic Co(II) ions on the model 56 amino acid residue protein, B1 immunoglobulin-binding domain of protein G, and successfully overcome the PCS averaging issues associated with the monodentate metal chelating probes. In addition, the same tags loaded with Cu(II) enable solid-state NMR PRE measurements to be performed to yield additional restraints on the protein structure.