Application of Relaxation Dispersion of Hyperpolarized ¹³C Spins to Protein–Ligand Binding

Abstract

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R 2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.

Competitive Displacement Restores the Hyperpolarized 15N NMR Signal in Blood Plasma

“…HP- 15 N as well as HP- 13 C labeled versions of drug molecules would be immensely useful to follow the in vivo metabolic fate of these compounds. However, the majority of drugs contain aromatic rings and/or nitrogen heterocycles, structural units that can strongly bind to albumin. , Interaction with albumin can speed up both the spin–lattice and spin–spin relaxation of NMR active nuclei present in these moleules. − Relaxometry of the aromatic protons of acetylsalicylate was used to study the interaction of this drug with albumin . Shortening of the T 1 and increased line width of the 13 C resonance in hyperpolarized [8- 13 C]­Aspirin (acetylsalicylic acid) due to binding and exchange with albumin was observed both in vitro and in vivo. , These undesirable effects were reduced somewhat in the presence of 3-fold excess of unlabeled Aspirin …”

SPICY: a method for single scan rotating frame relaxometry

“…62,[66][67][68][69] The single scan approach also significantly facilitates the use of nuclear spin hyperpolarization techniques [70][71][72][73] to boost the sensitivity of the experiments by several orders of magnitude. For example, recently Qi et al 74 boosted T 2 relaxation dispersion measurements by dissolution dynamic nuclear polarization (dDNP) to study protein-ligand binding. Similarly, SPICY could be exploited in hyperpolarized studies as well.…”

Ultrafast transverse relaxation exchange NMR spectroscopy

“…Furthermore, the single scan approach facilitates the use modern hyperpolarization methods to boost sensitivity by several orders of magnitude, allowing low concentration studies even in physiological concentrations, and the observation window can be extended by using heteronuclei such as 13 C with significantly longer T 1 relaxation time than 1 H. 36,38 The method can be exploited to study physical or chemical exchange phenomena in many disciplines ranging from chemistry and biochemistry materials science, including protein-ligand binding, rearrangement of secondary structures of RNA, metabolic processes and exchange phenomena related to novel biomarkers. 38,[49][50][51]…”