Internal motions in biomolecules studied by NMR spectroscopy

Since many biological processes occur on the μs to ms time scale, internal dynamics on that time scale is believed to 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 determination of exchange rates on that slow time scale. The formation of a small pheromone-protein complex is associated with some kind of internal rearrangements, necessary to accomodate the ligand. In this work, we are interested in the changes in internal dynamic upon the binding of a model odorant molecule, the 2-methoxy-3-isobutylpyrazine, to Major Urinary Protein I, MUP I. The study of the internal dynamics of both apo- and holo-MUP I has been carried out with the goal of monitoring the differences between the two states of the protein. In fact, impressive changes in chemical shifts are present between the two forms, indicating that some internal rearrangements have taken place. New and more classical experiments have been applied to MUP I in order to gain insight into the different types of motions that occurs at different time scales. The presence of chemical exchange has been identiτed in apo-MUP I using the classical Carr-Purcell-Meiboom-Gill experiment on single quantum 15N magnetisation. Surprisly, in holo-MUP I no presence of chemical exchange has been found, so in which time scale are the dynamics which leads to the release of the ligand in the environnment? Slow motions (ms-μs) in both apo- and holo-MUP I have been found using a new experiment, which highlight the presence of correlated motions in the backbone of proteins.

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

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

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

Puneet Ahuja

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.

EPFL2010