| dc.description.abstract | Nuclear magnetic resonance (NMR) spectroscopy is a widespread analytical technique used to characterize intermolecular interactions. However, due to the inherent insensitivity of NMR, long experimental times and large sample concentrations are required. Hyperpolarization methods are combined with NMR to broaden its application in characterizing intermolecular interactions rapidly. Here, hyperpolarization methods, based on Dissolution Dynamic Nuclear Polarization (D-DNP) and Para-Hydrogen Induced Polarization (PHIP) were developed to provide detailed interactions between proteins and small molecules.
D-DNP was used to characterize the interaction between hyperpolarized lipid molecules and an unfolded Outer membrane X (OmpX) under refolding conditions for the protein. Cross-relaxation rates between the different functional groups in the lipid and OmpX were determined in the absence of any denaturant. The fast experimental timescale of D-DNP allowed to access these conditions and may be useful for investigating structural changes in proteins during the refolding process. The PHIP technique requires minimal instrumentation and can be a cost-effective hyperpolarization technique for characterizing biomolecular interactions. Signal Amplification by Reversible Exchange (SABRE), the non-hydrogenative variant of the PHIP, allows renewal of polarization in solution. The pool of biological ligand motifs hyperpolarized by SABRE was broadened by developing a method, which allowed to hyperpolarize ortho-substituted N-heterocyclic molecules. Previously, these molecules yielded low polarization due to hindered binding to the SABRE catalyst. This steric hindrance was solved by adding smaller coligand molecules that allowed the formation of the polarization transfer complex. The incompatibility of the SABRE catalyst in water and proteins in alcohol required to develop a two-step approach involving flow-NMR for characterizing protein-ligand binding. The ligand was hyperpolarized in methanol, and subsequently mixed with protein to characterize binding interactions in a predominantly aqueous medium. Changes in the transverse relaxation rate (R₂) in the presence and absence of protein was monitored to identify binding. Thereafter, the hyperpolarization of a molecule acting as a reporter ligand was used in a competitive binding experiment to find dissociation constants (KD) that differ in three orders of magnitude for various ligands. Using a single reporter ligand allowed to determine KD of ligands not hyperpolarizable by SABRE. | |
