Hilty Research Group
Chemistry Department
Texas A&M University
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Investigating Biological Processes by Hyperpolarized NMR
We are exploiting dynamic nuclear polarization (DNP) for the development of new tools that
enable the rapid characterization of dynamic chemical processes. In the life sciences, these tools enable
the investigation of multiple aspects of protein-ligand interactions in drug discovery, along with the
fundamental characterization of the behavior of biological molecules, metabolites and proteins.
Hyperpolarized NMR of Protein-Ligand Interactions for Drug Discovery
Using dissolution dynamic nuclear polarization (DNP), in a single scan we routinely achieve a signal gain of three to four orders of magnitude when compared to conventional NMR. The technique of hyperpolarization has opened up an alternative way to study protein-ligand interactions. It enables not only the direct detection of ligand binding but also kinetics of ligand interactions with protein.
We have investigated competitive binding of two ligands to a protein by observing protein-mediated magnetization transfer between the two ligands. 1H spins of one of the two ligands are hyperpolarized and the fraction of this polarization induces NOE to the protein binding pocket which can be transferred to the second ligand. This effect is termed “hyperpolarized binding pocket NOE” (HYPER-BIPO-NOE). The enhanced signal intensities of the second ligand confirm that the two ligands competitively bind to a protein and also confirm the mode of binding of the second ligand due to the signal enhancement of specific protons. Moreover, relative signal build-up rates contain structural information on the binding epitope.
Fluorine-NMR spectroscopy has become a powerful tool to explore protein-ligand interactions benefited from 100% natural abundance of 19F. When DNP technique is applied to 19F-NMR, submicromolar concentrations of flourinated molecules are detectable which can benefit drug discovery. It is amenable to detect protein-ligand interactions in slow exchange which is limited by traditional NMR methods. We are developing techniques for the use of 19F-DNP to investigate ligand binding and binding dynamics. The dissociation constants for fluorinated ligands are calculated from a single one-dimensional spectrum which showed the ligand in free and bound states.
Probing Protein Folding in Real Time
Interactions that govern protein folding, one of the most fundamental biochemical processes, are often determined by NMR under equilibrium conditions. While highly specific to structure, such measurements do not directly monitor the folding process. However, using DNP hyperpolarization, signal enhancements in the liquid state of 2 to 3 orders of magnitude can routinely be obtained for 13C in polypeptides. This signal is sufficient to to study protein folding in real time while making use of the large chemical shift distribution of this nucleus. Proteins are hyperpolarized by dynamic nuclear polarization, and dissolved under denaturing conditions. The protein solution is then mixed with a different buffer to carry out a pH jump and trigger folding. Folding is monitored using a series of NMR experiments at different time points. Decomposing each transient into signal contributions from unfolded and folded protein fractions yields the folding rate constant. Additionally, resolved signals from individual sites carry specific information tied to the structure of the protein.
Metabolic Studies with High Resolution and Ultrahigh Sensitivity
High-resolution DNP-NMR enables a rapid analysis of small quantities of biological molecules. Using fractional isotope incorporation, it is further possible to determine metabolic pathways.
If biomolecules are synthesized by culturing microorganisms in a medium where a fraction of the carbon feedstock is labeled, coupling patterns observed in carbon spins in the synthesized molecule can be used to deduce the incorporation patterns of the carbon precursor. Additionally, the incorporation of atoms is also tracked by using partially labeled precursor molecules. The usage of a fractionally labeled feedstock also adds a quantitative dimension to dissolution DNP by enabling the comparison of the intensities of the observed multiplet to that of the singlet. The intensity ratios observed are indicative of the extent of label incorporated and is useful in determining the flux of the carbon precursor towards incorporation into the product molecule.
Hyperpolarized NMR of Protein-Ligand Interactions for Drug Discovery
Using dissolution dynamic nuclear polarization (DNP), in a single scan we routinely achieve a signal gain of three to four orders of magnitude when compared to conventional NMR. The technique of hyperpolarization has opened up an alternative way to study protein-ligand interactions. It enables not only the direct detection of ligand binding but also kinetics of ligand interactions with protein.
We have investigated competitive binding of two ligands to a protein by observing protein-mediated magnetization transfer between the two ligands. 1H spins of one of the two ligands are hyperpolarized and the fraction of this polarization induces NOE to the protein binding pocket which can be transferred to the second ligand. This effect is termed “hyperpolarized binding pocket NOE” (HYPER-BIPO-NOE). The enhanced signal intensities of the second ligand confirm that the two ligands competitively bind to a protein and also confirm the mode of binding of the second ligand due to the signal enhancement of specific protons. Moreover, relative signal build-up rates contain structural information on the binding epitope.
Fluorine-NMR spectroscopy has become a powerful tool to explore protein-ligand interactions benefited from 100% natural abundance of 19F. When DNP technique is applied to 19F-NMR, submicromolar concentrations of flourinated molecules are detectable which can benefit drug discovery. It is amenable to detect protein-ligand interactions in slow exchange which is limited by traditional NMR methods. We are developing techniques for the use of 19F-DNP to investigate ligand binding and binding dynamics. The dissociation constants for fluorinated ligands are calculated from a single one-dimensional spectrum which showed the ligand in free and bound states.
Probing Protein Folding in Real Time
Interactions that govern protein folding, one of the most fundamental biochemical processes, are often determined by NMR under equilibrium conditions. While highly specific to structure, such measurements do not directly monitor the folding process. However, using DNP hyperpolarization, signal enhancements in the liquid state of 2 to 3 orders of magnitude can routinely be obtained for 13C in polypeptides. This signal is sufficient to to study protein folding in real time while making use of the large chemical shift distribution of this nucleus. Proteins are hyperpolarized by dynamic nuclear polarization, and dissolved under denaturing conditions. The protein solution is then mixed with a different buffer to carry out a pH jump and trigger folding. Folding is monitored using a series of NMR experiments at different time points. Decomposing each transient into signal contributions from unfolded and folded protein fractions yields the folding rate constant. Additionally, resolved signals from individual sites carry specific information tied to the structure of the protein.
Metabolic Studies with High Resolution and Ultrahigh Sensitivity
High-resolution DNP-NMR enables a rapid analysis of small quantities of biological molecules. Using fractional isotope incorporation, it is further possible to determine metabolic pathways.
If biomolecules are synthesized by culturing microorganisms in a medium where a fraction of the carbon feedstock is labeled, coupling patterns observed in carbon spins in the synthesized molecule can be used to deduce the incorporation patterns of the carbon precursor. Additionally, the incorporation of atoms is also tracked by using partially labeled precursor molecules. The usage of a fractionally labeled feedstock also adds a quantitative dimension to dissolution DNP by enabling the comparison of the intensities of the observed multiplet to that of the singlet. The intensity ratios observed are indicative of the extent of label incorporated and is useful in determining the flux of the carbon precursor towards incorporation into the product molecule.