Screening of Protein-Ligand Binding Using a SABRE Hyperpolarized Reporter.

Hyperpolarization through signal amplification by reversible exchange (SABRE) provides a facile means to enhance nuclear magnetic resonance (NMR) signals of small molecules containing an N-heterocycle or other binding site for a polarization transfer catalyst. A purpose-designed reporter ligand, which is capable of binding both to a target protein and to the catalyst, makes the sensitivity enhancement by this technique compatible with the measurement of a range of biomolecular interactions. The H polarization of the reporter ligand 4-amidinopyridine, which is targeting trypsin, is used to screen ligands that are not themselves hyperpolarizable by SABRE.

Interfacing Liquid State Hyperpolarization Methods with NMR Instrumentation.

Advances in liquid state hyperpolarization methods have enabled new applications of high-resolution NMR spectroscopy. Utilizing strong signal enhancements from hyperpolarization allows performing NMR spectroscopy at low concentration, or with high time resolution. Making use of the high, but rapidly decaying hyperpolarization in the liquid state requires new techniques to interface hyperpolarization equipment with liquid state NMR spectrometers.

Characterization of protein-ligand interactions by SABRE.

Nuclear spin hyperpolarization through signal amplification by reversible exchange (SABRE), the non-hydrogenative version of -hydrogen induced polarization, is demonstrated to enhance sensitivity for the detection of biomacromolecular interactions. A target ligand for the enzyme trypsin includes the binding motif for the protein, and at a distant location a heterocyclic nitrogen atom for interacting with a SABRE polarization transfer catalyst. This molecule, 4-amidinopyridine, is hyperpolarized with 50% -hydrogen to yield enhancement values ranging from -87 and -34 in the and positions of the heterocyclic nitrogen, to -230 and -110, for different solution conditions.

2D NMR spectroscopy of refolding RNase Sa using polarization transfer from hyperpolarized water.

Polarization transfer from hyperpolarized water through proton exchange is used to enhance the NMR signals of amide protons of the Ribonuclease Sa protein. Spectra of the refolding protein are measured within 6 s after dilution of the denaturant urea, at urea-dependent folding rates adjusted in the range of 0.3-0.

Nuclear Spin Hyperpolarization of NH - and CH -Substituted Pyridine and Pyrimidine Moieties by SABRE.

Hyperpolarization of N-heterocycles with signal amplification by reversible exchange (SABRE) induces NMR sensitivity gains for biological molecules. Substitutions with functional groups, in particular in the ortho-position of the heterocycle, however, result in low polarization using a typical Ir catalyst with a bis-mesityl N-heterocyclic carbene ligand for SABRE, presumably due to steric hindrance. With the addition of allylamine or acetonitrile as coligands to the precatalyst chloro(1,5-cyclooctadiene)[4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene] iridium, the H signal enhancement increased in several substrates with ortho NH substitutions.

Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR.

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively.

Observation of Fast Two-Dimensional NMR Spectra during Protein Folding Using Polarization Transfer from Hyperpolarized Water.

Nuclear spin hyperpolarized water is utilized to obtain protein spectra not only in the folded state but also during the refolding process. Polarization transfer to through proton exchange and the nuclear Overhauser effect (NOE) results in NMR signal enhancements of amide protons by up to 24-fold. These enhancements enable the measurement of fast two-dimensional NMR spectra on the same time scale as the folding.