Protein–ligand binding interactions are characterized by the para-H2 based hyperpolarization technique SABRE and flow-NMR. Binding to the protein is identified by R2 change of a ligand first interacting with the Ir polarization transfer catalyst.
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 1H polarization of the reporter ligand 4-amidinopyridine,
which is targeting trypsin, is used to screen ligands that are not
themselves hyperpolarizable by SABRE. The respective protein–ligand
dissociation constants (K
D) are determined
by an observed change in the R
2 relaxation
rate of the reporter. A calculation of expected signal changes indicates
that the accessible ligand K
D values extend
over several orders of magnitude, while the concentrations of target
proteins and ligands can be reduced considering the sensitivity gains
from hyperpolarization. In general, the design of a single, weakly
binding ligand for a target protein enables the use of SABRE hyperpolarization
for ligand screening or other biophysical studies involving macromolecular
interactions.
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 Ribonuclease Sa 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. Resolved amide proton signals corresponding to the folded protein are observed both under folded and refolding conditions, whereby the refolding protein shows smaller transferred signals. Residue-specific evaluation of contributions to the polarization transfer indicates that signals attributed to a relayed intramolecular NOE are not observable in the refolding experiment. These differences are explained by the absence of long-range contacts and faster molecular motions in the unfolded protein. Applications of this method include accessing residue-specific information on structure and dynamics during multistate protein folding.
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 bismesityl 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 1 H signal enhancement increased in several substrates with ortho NH 2 substitutions. For example, for a proton in 2,4-diaminopyrimidine, the enhancement factors increased from À 7 � 1 to À 210 � 20 with allylamine or to À 160 � 10 with acetonitrile. CH 3 substituted molecules yielded maximum signal enhancements of À 25 � 7 with acetonitrile addition, which is considerably less than the corresponding NH 2 substituted molecules, despite exhibiting similar steric size. With the more electron-donating NH 2 substitution resulting in greater enhancement, it is concluded that steric hindrance is not the only dominant factor in determining the polarizability of the CH 3 substituted compounds. The addition of allylamine increased the signal enhancement for the 290 Da trimethoprim, a molecule with a 2,4-diaminopyrimidine moiety serving as an antibacterial agent, to À 70.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.