Applications of Long-Lived Spin States to High Resolution NMR Studies of Biomolecules

Slow dynamic processes, such as biomolecular folding/unfolding, macromolecular diffusion, etc., can be conveniently monitored by solution-state two-dimensional (2D) NMR spectroscopy, provided the inverse of their rate constants does not exceed the nuclear spin-lattice relaxation time constants (T1). The discovery of long-lived states (LLS) by Malcolm Levitt's group opened a new dimension for the study of slow dynamic phenomena, as magnetization stored in the form of LLS decays with the time constants TLLS, where in many cases TLLS >> T1. In this thesis, various excitation methods and applications of LLS are discussed. Broadband excitation of LLS is suitable to monitor slow processes and has been applied to study the slow ring-flip in tyrosine residues of BPTI (Bovine Pancreatic Trypsin Inhibitor), as well as to perform simultaneous measurements of diffusion coefficients in mixtures of molecules with arbitrary J-couplings and chemical shifts. The applications of LLS, initially believed to be limited to isolated spin-½ pairs, were extended to larger spin systems, including some common amino acids like Serine, Aspartic Acid, etc. LLS have been observed in Glycine residues of small peptides like Ala-Gly, as well as in mobile parts of proteins, e.g., in Gly 75 and 76 of Ubiquitin. The lifetimes TLLS are more sensitive to dipolar interactions with external spins than longitudinal and transverse relaxation time constants, T1 or T2, and therefore can provide structural information for unfolded proteins. The unfolding of Ubiquitin by addition of Urea and by varying pH was followed using LLS. The excitation of coherent superpositions across singlet and triplet states, which we call long-lived coherences (LLC's), leads to resolution enhancement in conventional NMR spectroscopy. New methods have been designed to store hyperpolarized (13C or 1H) magnetization in the form of LLS and have been demonstrated using samples of Ala-Gly and Acrylic Acid.

Measurement of cross-correlation of fluctuations of dipolar couplings and anisotropic chemical shifts by selective spin locking

Geoffrey Bodenhausen, Robert Konrat

New exptl. methods are presented that allow one to measure cross-correlation between the fluctuations of dipolar interactions and chem. shift anisotropy (CSA) in scalar coupled systems with two and three spins I = 1/2 in isotropic soln. The methods involve selective excitation of the transverse magnetization IAx of a chosen spin A, followed by selective spin locking of this magnetization. If the spin locking radiofrequency (RF) field is exactly on-resonance for spin A while leaving the other spins M and X unaffected, there is no cross-relaxation (Overhauser effect) in the rotating frame, i.e., no transfer from IAx to IxM or IXx. By contrast, cross-correlation between different relaxation mechanisms may give rise to single-quantum coherence of higher rank. In particular, CSA-dipolar cross-correlation may lead to a partial conversion of IAx into 2IAxIMz or 2IAxIXz, while interference between two dipole-dipole interactions leads to the appearance of terms such as 3IAxIMzIXz. [on SciFinder (R)]

1992

Coherence transfer in nuclear magnetic resonance by selective homonuclear Hartmann-Hahn correlation spectroscopy

Geoffrey Bodenhausen, Robert Konrat

New NMR techniques are described for transferring transverse magnetization between scalar-coupled nuclei in the rotating frame. The methods involve simultaneous spin-locking of two selected spins A and X, using cosinusoidal modulation of the radio frequency (rf) carrier. If transverse magnetization of spin A is excited selectively before spin-locking, for example with a self-refocusing 270 Deg Gaussian pulse, in-phase magnetization will be transferred from spin A to spin X, and in-phase multiplets are obtained in both one- and two-dimensional spectra. The method allows one to verify whether two particular spins are connected by a scalar coupling. Examples are shown for peptides and basic pancreatic trypsin inhibitor (BPTI). The fine structure of the multiplets yields information on couplings to further spins. In contrast to nonselective spin-lock expts., the magnetization cannot diffuse to a manifold of coupling partners. If relayed coherence transfer is to be achieved deliberately, several spins may be locked simultaneously by imposing an addnl. modulation on the rf carrier. [on SciFinder (R)]

1991

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