Detecting the Undetectable: Functional Protein Motions in the Hidden Timescale Window Revealed by NMR Relaxation Measurements

Abstract

About 50 years ago, even before NMR was ready to determine structure of proteins, it could unveil a surprising motion in aromatic side-chain (Wuthrich & Wagner, 1975) from 1D 1H spectra. With decades of development, NMR is now the most powerful technique for studying atomic resolution dynamics in proteins at biological conditions, even inside live cells. Although NMR offers relaxation-based methods to determine motion on the entire dynamics timescale spectrum, accurate characterization of the supra-τc dynamics (4 ns-40 µs) was not possible due to technical limitations. Dynamics from this timescale window is indicated to play a decisive role in molecular recognition and binding events. By combining the advancement in hardware with smart design of pulse programs, we have developed a high-power RD (relaxation dispersion) method, which can accurately detect dynamics in this previously inaccessible window. By applying this newly developed high-power R1ρ RD, a hidden supra-τc motion was found in the first loop of the well-known protein GB3. From the timescale of motion measured at several supercooled temperatures, the activation energy for the loop-motion was estimated to be 65.6 kJ mol-1. Arrhenius extrapolation showed that the loop moves with a timescale of ~400 ns at physiological temperature of 308 K. Analyzing the 640-membered ERMD (Ensemble-Restrained-Molecular-Dynamics) RDC ensemble, we found elevated dynamics from higher fluctuation of backbone atoms as well as lower supra-τc order parameters in the region where RD is detected. Interestingly, the newly observed supra-τc motion takes place in a region, which binds to antibodies. This hints to a link of the observed motion with the antibody recognition of GB3. After unveiling a functional backbone motion in GB3, in chapter 2 we have studied the dynamics of methyl groups in the side-chains of ubiquitin, with a new type of RD method; 13C Extreme-CPMG (E-CPMG), developed in our group. This method can cover the detectable timescale range of both conventional CPMG and R1ρ. E-CPMG reported the same timescale and amplitude of motion, which was previously found from R1ρ measurements with much longer measurements time. Similarity in timescale and activation energy of the detected side-chain motion with previously found backbone dynamics hints towards a common mechanism. This hypothesis was proven by the absence of the side-chain motion in two different single point mutants (E24A and G53A), which were designed and tested for the quenching of backbone dynamics. In chapter 3, we have extended the E-CPMG approach to a nucleus (1H) with higher gyromagnetic ratio, where we could generate higher B1 field with less applied RF power. We found and corrected a linear decay in RD profiles, arising from the phase cycle, which is widely used even in conventional CPMG measurements. Using this approach, we could detect the peptide-flip induced breathing motion in twice as many residues in ubiquitin compared to a previous report. In addition, we could directly detect the large amplitude pincer-mode motion for the first time, in segments where the existence of supra-τc motion was predicted from both RDC and MD simulations. This newly detected motion was already predicted to contribute to the conformational adaption power of ubiquitin while binding to other proteins. Finally, in chapter 4, we found a very fast (4 µs) dynamics at 263 K in an intrinsically disordered protein p53-TAD, in residues which are found to be in helical conformation in a bound complex. So far, this is the fastest detected motion with RD. The helical propensity was also predicted in the same stretch of residues from SSP (secondary structural propensity) score calculation with the experimental chemical shifts of the free protein. A great reduction in exchange rate (~100 times) was found when the proline residue at the C-terminal of the helix was removed. In addition to that, more residues, including some from a second helix, were found to undergo conformational exchange. A doubling in helical propensity was found in this mutant (P27A) from SSP score calculations. In two other mutants (W23A; F19A and W23A; F19A; P27A) where the hydrophobic residues with aromatic side-chains were removed, no conformational exchange was detected. These finding suggests that the RD-observed 4 µs motion could originate from fast folding of the transiently formed helix which is assisted by the hydrophobic core in the center. In this thesis, by applying high-power RD, functionally relevant supra-τc motion is discovered in both backbone and side-chains of well-folded and disordered proteins. These findings helped in understanding molecular recognition and folding processes in the studied proteins.