Solution NMR of Polypeptides Hyperpolarized by Dynamic Nuclear Polarization

Abstract

Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300-2000 are obtained in partially deuterated polypeptide when hyperpolarized on (13)C and of 30-180 when hyperpolarized on (1)H. A simulated spectrum is used to identify different resonances in the hyperpolarized (13)C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.

Rapid-melt Dynamic Nuclear Polarization

“…Other applications include biomolecular NMR [63,64], biological assays [65,66], detection of reaction intermediates having low concentrations [67], protein folding [68], and drug screening [69]. The use of dissolution DNP in a clinical environment is under development [70].…”

“…Other uses of dissolution DNP include biomolecular NMR [63,64], the study of biological assays [65,66] and the detection of low concentration reaction intermediates [67]. Only a few analytical application have been demonstrated [120].…”

“…Details of these methods can be found elsewhere [91,92]. Dissolution DNP is used for metabolic imaging, biomolecular NMR [63,64] and biological assays [65,66] while MAS-DNP has found its applications in surface chemistry, protein structure determination and polymer studies [41,40]. Dissolution DNP is limited to slow relaxing nuclei and only a few number of scans are possible within the T 1 of the nuclei of interest [70].…”

Nuclear spin relaxation in liquids and gases

“…The same group investigated also the influence of Gd(III) doping on the DNP in the systems containing pyruvate ( 13 C labelled in position 1) and various nitroxides. 165 Ragavan and co-workers 166 investigated the possibility to apply the dissolution DNP to a uniformly 13 C-labelled, 50% deuterated polypeptide. The polypeptide sample was polarized in native and denaturing conditions.…”

Simultaneous estimation of T1 and the flip angle in hyperpolarized NMR experiments using acquisition at non-regular time intervals

“…Through electron-nuclear dipolar interaction the perturbation of the Boltzmann distributions enhances the net magnetization $10,000 fold [6]. To date, DNP has been used to characterize inorganic compounds [7,8], study pharmacokinetics [9], and determine structures of solid materials [10][11][12][13][14], solvents [15,16], polypeptides [17,18], lipids [19,20], and to monitor the biodistribution or metabolism of urea [6,[21][22][23], pyruvate [22,[24][25][26][27], and other even more complex metabolites [28,29].…”