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.
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