Developing new paramagnetic NMR method to study protein dynamics

Discover the ultra-weak interaction between proteins and its significance through PRE

Using paramagnetic NMR spectroscopy, we found a weak non-covalent interaction between ubiquitin monomers (KD≈5 mM), and analyzed the dimer ensemble structure. We further visualized the active-state complex with an estimated lifetime in microseconds. High in local effective concentration, short-lived fleeting protein-protein interactions (KD≈25 mM) would be essential for the cell signal to propagate.
Reference:   Angew Chem Int Ed, 2012, 51, 469-472.
                    Angew Chem Int Ed, 2014, 53, 11501-11505.

 Protein Structural Ensembles Visualized by Solvent Paramagnetic Relaxation Enhancement

We have synthesized a new sPRE probe, Gd(III)–TTHA–TMA, and developed label-free paramagnetic NMR technology (solvent PRE). The small molecule probe has become an objective “observer”, can be accurate and convenient access the structure information of proteins in solution. Based on this principle and integrate molecular dynamics simulation sampling and Monte Carlo selection process, the new method can reproduce the dynamic distribution of protein in solution
Reference: J Biomol NMR, 2014, 58, 149-154.
                   Angew Chem Int Ed, 2017, 56,1002-1006.  

PRE NMR capture multiple conformational states of K63-diUb

We combined paramagnetic relaxation enhancement (PRE) with small angle X-ray scattering (SAXS) demonstrate that K63-diUb populates considerable closed conformation. The further analysis indicated that closed conformation can be divided into at least two groups to accommodate different target proteins. We uncovered the high-resolution closed conformation K63-diUb adopted in solution and revealed that the mechanism of binding is conformational selection. 
Reference: eLife, 2015, 4:e05767. 

Using solvent PRE to characterize protein conformational dynamics

We have developed the sPRE calculation method. The sPRE can be computed with a stand-alone program for rapid evaluation, or with the invocation of a module (PSolPot) in the latest release of the structure calculation software Xplor-NIH. We demonstrate the sPRE method using GB1, a structurally rigid protein, and calmodulin, a protein comprising two domains and existing in open and closed states.
 Reference:  Methods, 2018, 148:48-56.

Designing a rigid PRE probe to visualize the subtle dynamics of holo QBP protein

We designed a Cu(II)-based paramagnetic probe, with the metal ion sandwiched between a di-histidine motif engineered at a protein helix and a small capping molecule. Using this rigid paramagnetic probe, we observed subtle dynamic fluctuation (~1 Å) for a loop in the holo QBP. Elucidation of subtle protein dynamics would afford a better understanding of the mechanism for protein function.
Reference: Biochemistry, 2014,53, 1403-1409.

Lanthanoid tagging via an unnatural amino acid for protein structure characterization

Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags.
Reference: J Biomol NMR, 2017, 67:273-282.