Long lived states in high field NMR spectroscopy for the study of very slow dynamic processes in solution state

In solution-state NMR spectroscopy, dynamic processes can be probed provided the nuclear relaxation rate constant does not exceed the rate constant of the process. Phenomena occurring on a time scale longer than the spin-lattice relaxation time constant, T1, were not amenable to study by conventional NMR. The discovery of long-lived nuclear spin states (LLS) in 2004, by M. Levitt, M. Carravetta and O. W. Johannessen, showed that lifetimes of magnetization can be much higher than T1. LLS experiments can be performed either in zero or in high magnetic fields for scalar-coupled proton spin pairs. This thesis contributes methods for exciting and preserving LLS in high field for a wide class of molecules, including sugars, amino acids and nucleotides. Slow diffusion has been measured for a mixture of molecules having different NMR parameters using LLS. Conformational exchange could also be followed by exchange spectroscopy in BPTI. Recently, LLS have been observed in highly mobile parts (Gly-75 and 76) of Ubiquitin. Dynamic Nuclear Polarization (DNP) is one of the methods to overcome the inherent low sensitivity of NMR spectroscopy. We have conceived an experiment to preserve DNP enhanced magnetization by conversion into LLS. A way of improving resolution and sensitivity of NMR has been designed by creation of long-lived coherences (LLC) in biomolecules.

Methods for nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) measurements using long-lived states

Geoffrey Bodenhausen, Arnaud Comment, Sami Jannin, Riddhiman Sarkar, Paul Romeo Vasos

A method for nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) measurements, includes creation of enhanced polarization of nuclei of a first kind within a sample in a magnetic field at cryogenic temperatures and transfer of the polarized sample to room temperature. The enhanced polarization of nuclei of the first kind is thereby transformed into long-lived states (LLS) of nuclei of a second kind and these LLS are sustained. The LLS is at least partially converted into observable magnetization and an NMR or MRI measurement is carried out. The method allows one to extend the time needed between hyperpolarized magnetization and NMR detection.

2013

Measurement of cross relaxation between two selected nuclei by synchronous nutation of magnetization in nuclear magnetic resonance

Geoffrey Bodenhausen, Robert Konrat

A novel technique is described that allows one to measure cross-relaxation rates (Overhauser effects) between 2 selected nuclei in high-resoln. NMR. The 2 chosen sites are irradiated simultaneously with the sidebands of an amplitude-modulated radiofrequency field, so that their magnetization vectors are forced to undergo a simultaneous motion, which is referred to as synchronous nutation. From the time-dependence obsd. for different initial conditions, one may derive cross-relaxation rates, and hence det. internuclear distances. The scalar interactions between the selected spins and other spins belonging to the same coupling network are effectively decoupled. Also, cross relaxation to other spins in the environment does not affect the transient response of the selected spins, which are therefore in effect isolated from their environment in terms of dipolar interactions. The method is particularly suitable to study cases where normal Overhauser effects are perturbed by spin-diffusion effects due to the presence of further spins. The technique is applied to the protein bovine pancreatic trypsin inhibitor. [on SciFinder (R)]

1993

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful and informative methods to probe molecular dynamics. Specifically, chemical exchange is an important phenomenon and one of the earliest and most vigorously investigated in NMR spectroscopy. Its effects can be observed in many NMR spectra; lines in a spectrum are broadened or averaged because the nuclei are exchanging magnetic environments. A wide variety of experimental methods exists to determine exchange rates of protons that hop between amino acids and water in the slow or intermediate exchange regime. However, so far there is a lack of methods to investigate hydrogen exchange in the fast exchange regime. In this thesis, we focus on the determination of very fast chemical exchange rates from the effects of scalar relaxation on a coupled nucleus. We quantify the effects of scalar relaxation caused by exchanging hydrogens, by detecting the decay of a 15N coherence under a multiple-refocusing Carr-Purcell-Meiboom-Gill (CPMG) pulse train in the presence or absence of hydrogen decoupling. In particular, we have adapted to the case of deuterium a method that was originally designed to study the exchange rate of the indole proton of tryptophan in water as a function of pH and temperature. We will present the exchange rates of the indole deuterium in tryptophan with solvent heavy water. After that, the proton exchanges rates have been compared with the deuterium exchange rates in order to describe the kinetic isotope effect. In a similar manner, we have quantified the proton exchange rates in pure water by using multiple refocusing of transverse 17O magnetization as a function of pH and temperature. Such measurements are widely used to obtain valuable information about molecular dynamics and structure of proteins, protein complexes and nucleic acids. The knowledge of the kinetic isotope effect may contribute to the characterization of reaction mechanisms and other dynamic parameters that can give insight into the stability of hydrogen bonded secondary structures in biomolecules. This information could form a foundation for a more thorough theoretical or modeling development of a number of long and outstanding fundamental questions. In the second part of the thesis, we combine fast exchange with dissolution dynamic nuclear polarization (D-DNP) to study intrinsically disordered proteins (IDPs). IDPs constitute a very large and functionally important class of proteins that lack stable secondary and tertiary structures. As a consequence of the broad dynamics and flexibility of IDPs, and specially under physiological conditions, the NMR spectra of IDPs typically show a strong signal overlap and broadening due to proton exchange. We present a method to overcome these limitations by means of hyperpolarized HDO produced by D-DNP. We could study the effects of conformational adaptations in Osteopontin (OPN), a metastasis- associated intrinsically disordered protein (IDP) by chemical exchange. The magnetization from hyperpolarized water is transferred to the rapidly exchanging protons of the solvent-exposed residues of the protein. The exchange with hyperpolarized water boosts the signals of fast exchanging residues above the detection limits. This allows one to follow individual resonances and draw conclusions regarding conformational adaptations due to ligand binding under physiological conditions. Thus, with our approach, exchange with the solvent is turned into an advantage

EPFL2017

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

EPFL2017