Chemical exchange in proteins | EPFL Graph Search

Nuclear Magnetic Resonance (NMR) spectroscopy offers many ways to investigate dynamic properties of molecules. A wide variety of experimental techniques can probe molecular dynamics on time scales that range from 10-12 to 103 seconds. Many lines of evidence point to the existence of large scale dynamics on milli- to microsecond time scale that are essential to molecular function. Those conformational motions can give rise to chemical exchange effects. So far in the literature two methods have been mainly used to study such processes: CPMG echo trains and R1ρ spin-lock relaxation experiments. The former is suited to investigate processes on the millisecond time scale, whereas R1ρ can probe processes ranging from milliseconds down to ca. microseconds. CPMG and R1ρ studies of the relaxation rates of single quantum (SQ) coherences can be complemented by multiple quantum coherence (MQ) coherences experiments which can provide information whether two (or more) spins are affected simultaneously by chemical exchange. The goal of this thesis is to characterize chemical exchange in proteins by new (Heteronuclear Double Resonance, HDR) and existing (relaxation compensated CPMG) methods. The presence of chemical exchange has been identified in APO-rMUP using the classical CPMG experiment on single quantum 15N magnetization, whereas in HOLO-rMUP no presence of chemical exchange has been found. Most of the thesis is dedicated to the analysis and the application of a new method, HDR, which is designed to determine the cross-relaxation rates between MQ operators. In fact, in heteronuclear systems, correlated chemical exchange contributes to cross-relaxation between MQ coherences 2IxSx and 2IySy. This method, based on MLEV-32 and WALTZ-16 used in double resonance mode, is designed to effectively preserve all relevant MQ coherences simultaneously so that the interconversion between MQ operators can only arise through cross-relaxation. This is an essential requirement if we want to measure, for example, small cross-relaxation rates between MQ operators. We have treated the effects of coherent evolution during the HDR sequences by numerical simulations and Average Hamiltonian Theory (AHT) showing that, under most conditions, the dynamics of MQ coherences is not affected by coherent evolution. This result is proved experimentally on the 15N-1H pair of selectively deuterated tBoc-glycine. The effect of relaxation during HDR sequences leads to an effective relaxation superoperator which is explored by numerical simulations and predicted by Average Liouvillian Theory (ALT). Finally the HDR MLEV-32 sequence is applied to amide 1H-15N pairs in ubiquitin and KIX proteins. In the case of ubiquitin the HDR MLEV-32 experiments and the numerical simulations run with parameters obtained from the literature are in good agreement, suggesting that HDR sequences could be used as a tool to obtain information about the dynamics of the system.

Improving Sensitivity in Solid State NMR

Sensitivity in Nuclear Magnetic Resonance (NMR), especially in solid-state NMR, has always been a challenging and important issue and thus a motivation for new developments. The magnetic field B0, the gyromagnetic ratios of the observed nuclei, as well as the preparation of the samples are important for optimizing the sensitivity. Often only a small amount of sample is available and the nuclei of interest have low gyromagnetic ratios, which makes solid-state NMR extremely time consuming and, in some cases, practically impossible. In addition, strong interactions can cause extensive line-broadening, thus making it difficult to excite or detect high resolution spectra. In this thesis two main aspects concerning sensitivity in solid-state NMR are addressed. The first part reveals the difficulties of the excitation and detection of quadrupolar nuclei, in particular of the highly abundant nucleus nitrogen-14, and presents an indirect detection method combined with a more efficient excitation and reconversion scheme using Delays Alternating with Nutations for Tailored Excitation (DANTE). The second part presents a new experimental set-up for Dynamic Nuclear Polarization (DNP), which has evolved into a very successful method at the Ecole Polytechnique Fédérale de Lausanne. It is shown in this work that the DNP method combined with solid-state NMR allows one to enhance NMR signal intensities by one or two orders of magnitude, thus opening the doors towards a great variety of new exciting applications. In solids spinning at the magic angle, the indirect detection of single-quantum (SQ) and double-quantum (DQ) 14N spectra (I = 1) via spy nuclei S = 1/2 such as protons can be achieved in the manner of heteronuclear single- or multiple-quantum correlation (HSQC or HMQC) spectroscopy. The coherence transfer is achieved by recoupling the heteronuclear dipolar interactions, i.e., with symmetry-based sequences. The line broadening caused by the homogeneous decay of the transverse terms of the spy nuclei S can be reduced by implementing suitable dipolar decoupling schemes during the evolution period. The lack of efficiency of the excitation and reconversion can be overcome by replacing the conventional rectangular pulses applied to the nitrogen-14 nuclei by a train of short rotor-synchronized pulses in the manner of DANTE. These pulse trains excite a large number of crystallites in the sample uniformly and allow one to efficiently excite magnetization over large bandwidths, limited only by the quality factor of the probe. When combining N interleaved DANTE schemes, obtained by applying N pulses per rotor period, leading to so-called "DANTE-N" sequences, one obtains methods that greatly improve the uniformity of the excitation and lead to higher signal intensity in less rotor periods than basic DANTE. Applications to nitrogen-14 with fast magic angle spinning (typically νrot ≥ 60 kHz), using either direct or indirect detection, are backed up by simulations that provide insight into the properties of basic and interleaved DANTE sequences. DNP exploits the polarization transfer, driven by microwave irradiation, from unpaired electrons, usually carried by polarization agents such as TEMPO or TOTAPOL, to the target nuclei. This transfer results in enhancements of the signal-to-noise ratios and thus opens many new possibilities for challenging samples. In this thesis the experimental requirements are demonstrated and important parameters like the temperature dependence are investigated. Various sample preparation methods are proposed, e.g., by attaching the radical directly to the molecules under investigation, instead of preparing a solution that should forma glass upon freezing. The combination of indirect detection of 14N with DNP improves the signal-to-noise ratio dramatically and shortens the recovery delay between subsequent experiments. Besides samples of biological interest, it was also shown that the NMR signals of molecules on surfaces of various materials can be greatly enhanced by DNP. A characterization of the distribution of surface binding modes and interactions in a series of functionalized materials could be demonstrated. The bonding topology of functional groups in materials obtained via a sol-gel process and in materials prepared by post-grafting reactions could be identified and compared. Furthermore, the remarkable gain in time afforded by DNP allows the facile acquisition of two-dimensional correlation spectra. The surfaces were characterized by transferring the polarization from protons to other nuclei such as 13C, 29Si and 27Al by means of cross-polarization.

EPFL2012

Dynamic Studies through Control of Relaxation in NMR Spectroscopy

Nicola Salvi

In numerous biological processes that constitute the base of living organisms, protein function is fundamentally related to internal dynamics occurring on μs-ms time scales that can give rise to chemical exchange contributions to relaxation. In a heteronuclear two-spin system (e.g., 1H-15N), correlated motions of the two nuclei induce cross-relaxation between multiple-quantum coherences that can be quantified using new Heteronuclear Double Resonance (HDR) techniques. An analytical model describes the effect of an applied radio-frequency field on the relaxation rate of interest in the presence of fast exchange, providing accurate information on the kinetics of correlated processes. Numerical simulations and experimental results confirm the validity of the model. HDR and more conventional relaxation dispersion techniques are used to characterize the internal dynamics in several biological systems. Motions on similar time scales are detected in the two different binding surfaces in human ubiquitin and in the linker between them (Phe45), suggesting the presence of a possible global motion. Additional applications of HDR give insights into the dynamics occurring in the KID-binding domain (KIX) of the CREB-binding protein. We identified the presence of exchange processes, faster than the one reported by Konrat and coworkers, outside the main helices of KIX. These fast motions are most likely the sign of conformational disorder within the native state, which may promote the transition to the unfolded ensemble. A combination of 15N relaxation rates and magnetization transfer rates are used to provide a qualitative characterization of internal dynamics in Engrailed 2. While contribution of exchange processes in the ms time scale are small, fluctuations at sub-ms timescales are found to occur both in the unstructured part that contains important binding sites for other transcription factors, and at a few restricted locations in the structured homeodomain. Such motions are characterized by 15N R1ρ relaxation dispersion. Our results reveal that the hexapeptide motif is characterized by complex dynamics that may be linked to its physiological function. Moreover, the time scale of motions in the homeodomain is reasonably close to that of the hexapeptide region. It is therefore tempting to suppose that transient contacts occur between the structured and unstructured regions.

EPFL2013

Preservation of heteronuclear multiple-quantum coherences in NMR by double-resonance irradiation

Geoffrey Bodenhausen, Fabien Ferrage, Simone Ulzega, Mariachiara Verde

A heteronuclear double-resonance (HDR) method based on MLEV-32 or WALTZ-32 pulse sequences has been designed for the investigation of relaxation of heteronuclear multiple-quantum (MQ) coherences. The theoretical analysis of this technique uses average Hamiltonian theory (AHT) to treat the effects of coherent evolution associated with scalar couplings, offsets, and inhomogeneous radiofrequency (rf) fields during the pulse sequence. Under most conditions, the dynamics of the MQ coherences during the HDR sequence is not affected by rf inhomogeneities and scalar couplings for offsets as large as the nutation frequency. The predictions drawn from AHT are supported by numerical simulations and experiments.

2009

Improving Sensitivity in Solid State NMR

EPFL2012

Dynamic Studies through Control of Relaxation in NMR Spectroscopy

Nicola Salvi

EPFL2013