Dynamic Studies through Control of Relaxation in NMR Spectroscopy

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.

Chemical exchange in proteins

Mariachiara Verde

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.

EPFL2009

Slow dynamics in biomolecules studied by NMR spectroscopy

Julien Wist

Since many biological processes occur on the μs to ms time scale, internal dynamics on that time scale may well be related to biological functionality. The characterization of internal dynamics is thus an important issue to improve our understanding of the biological activity of macromolecules, such as proteins, RNA and DNA fragments. Solution NMR spectroscopy, in particular relaxation measurements, is well suited for the deter tion of exchange rates on that slow time scale. In solution, relaxation of nuclear spins is caused mainly by Brownian tumbling and, to a lesser extent, by internal motions that modulate the effective field of the spins. Thus, these fluctuations depend on the electronic environment of each spin and on internal dynamics. On the one hand, the determination of structures of biomolecules suffers from local motions that lead to the determination of average structures. While, on the other hand, only little information is available concerning the time scale and the type of motions that may be responsible for important biological functions. To address this issue, new experiments have been designed to determine the contribution of slow internal motions (μs-ms) to relaxation of multiple-quantum coherences (MQC) involving carbonyl C' and nitrogen N nuclei. Local motions that are slower than the overall correlation time τc (ns) induce chemical shift modulations (CSM) of the spins involved in the motions. If correlated, such fluctuations can contribute to differential relaxation between zero- and double-quantum coherences. It is known that a train of π-pulses can reduce this contribution if the time scale of the modulations is comparable to the frequency at which refocusing pulses are applied. In the limit of slow pulse rates. both CSM/CSM (slow dynamics) and CSA/CSA (structure), the sum of this two terms being referred to as CS/CS, affect the differential relaxation of ZQC and DQC, whereas in the limit of fast pulse rates, only CSA/CSA affect the differential line broadening, allowing the discrimination between structural and dynamic contributions to relaxation. Related theoretical concepts and experimental methodology are extensively discussed in the text. In protonated and deuterated ubiquitin it was possible to confirm that the particularly large C'/N cross-correlated CS/CS relaxation rates observed for the Asparagine N25 residue were partly caused by a pronounced local mobility. This is in agreement with previous observations that indicate mobility in this part of the protein: the fact that the nitrogen R2 relaxation rate of N25 deviates from the N/NHN CSA/DD cross-correlated relaxation rates, the fact that the neighbouring Glutamic acid residue E24 is almost absent from the HSQC spectrum and that Isoleucine I23 shows a particularly large N/HN cross-correlated CS/CS relaxation rate.

EPFL2005

Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

Geoffrey Bodenhausen, Fabien Ferrage, Nicola Salvi, Simone Ulzega

Understanding how proteins function at the atomic level relies in part on a detailed characterization of their dynamics. Ubiquitin, a small single-domain protein, displays rich dynamic properties over a wide range of time scales. In particular, several regions of ubiquitin show the signature of chemical exchange, including the hydrophobic patch and the beta 4-alpha 2 loop, which are both involved in many interactions. Here, we use multiple-quantum relaxation techniques to identify the extent of chemical exchange in ubiquitin. We employ our recently developed heteronuclear double resonance method to determine the time scales of motions that give rise to chemical exchange. Dispersion profiles are obtained for the backbone NHN pairs of several residues in the hydrophobic patch and the beta 4-alpha 2 loop, as well as the C-terminus of helix alpha 1. We show that a single time scale (ca. 50 mu s) can be used to fit the data for most residues. Potential mechanisms for the propagation of motions and the possible extent of correlation of these motions are discussed.