Kinetics of Conformational Sampling in Ubiquitin (original) (raw)
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Proceedings of the National Academy of Sciences, 2006
Single-molecule fluorescence experiments have shown that the conformation of the complex between Escherichia coli general NAD(P)H:flavin oxidoreductase (FRE) and flavin adenine dinucleotide (FAD) fluctuates over a range of timescales between 10 −4 and 1 s. Here we use 15 N and 13 C relaxation dispersion NMR methods to study millisecond-timescale dynamics in the complex. In this time regime, the protein is extremely flexible, with residues that undergo conformational exchange located throughout the molecule. Three distinct regions of dynamics are quantified, with two of them involving residues making contact to the donor (Tyr-35) and acceptor (FAD) sites that participate in the electron transfer reaction monitored in single-molecule experiments. Modulation of the donor–acceptor distance through these conformational exchange processes, occurring with rates of ≈400 and 1,200 s −1 (22°C), affects the rate of electron transfer and partially accounts for the range of the observed dynamics...
Selective Characterization of Microsecond Motions in Proteins by NMR Relaxation
Journal of the American Chemical Society, 2009
The three-dimensional structures of macromolecules fluctuate over a wide range of timescales. Separating the individual dynamic processes according to frequency is of importance in relating protein motions to biological function and stability. We present here a general NMR method for the specific characterization of microsecond motions at backbone positions in proteins even in the presence of other dynamics such as large-amplitude nanosecond motions and millisecond chemical exchange processes. The method is based on measurement of relaxation rates of four bilinear coherences and relies on the ability of strong continuous radio frequency fields to quench millisecond chemical exchange. The utility of the methodology is demonstrated and validated through two specific examples focusing on the thermo-stable proteins, ubiquitin and protein L, where it is found that small-amplitude microsecond dynamics are more pervasive than previously thought. Specifically, these motions are localized to α helices, loop regions, and regions along the rim of β sheets in both of the proteins examined. A third example focuses on a 28 kDa ternary complex of the chaperone Chz1 and the histones H2A.Z/H2B, where it is established that pervasive microsecond motions are localized to a region of the chaperone that is important for stabilizing the complex. It is further shown that these motions can be well separated from extensive millisecond dynamics that are also present and that derive from exchange of Chz1 between bound and free states. The methodology is straightforward to implement, and data recorded at only a single static magnetic field are required.
Journal of Biomolecular NMR, 2013
Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps-ls motions in proteins by solid-state NMR.
Molecules, 2013
Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool that has enabled experimentalists to characterize molecular dynamics and kinetics spanning a wide range of timescales from picoseconds to days. This review focuses on addressing the previously inaccessible supra-τ c window (defined as τ c < supra-τ c < 40 s; in which τ c is the overall tumbling time of a molecule) from the perspective of local inter-nuclear vector dynamics extracted from residual dipolar couplings (RDCs) and from the perspective of conformational exchange captured by relaxation dispersion measurements (RD). The goal of the first section is to present a detailed analysis of how to extract protein dynamics encoded in RDCs and how to relate this information to protein functionality within the previously inaccessible supra-τ c window. In the second section, the current state of the art for RD is analyzed, as well as the considerable progress toward pushing the sensitivity of RD further into the supra-τ c scale by up to a factor of two (motion up to 25 s). From the data obtained with these techniques and methodology, the importance of the supra-τ c scale for protein function and molecular recognition is becoming increasingly clearer as the connection between motion on the supra-τ c scale and protein functionality from the experimental side is further strengthened with results from molecular dynamics simulations.
Journal of the American Chemical Society, 2012
We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr−Purcell−Meiboom−Gill relaxationdispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.
ChemPhysChem, 2018
Studying protein dynamics on microsecond-to-millisecond (μs-ms) time scales can provide important insight into protein function. In magic-angle-spinning (MAS) NMR, μs dynamics can be visualized by R 1ρ rotating-frame relaxation dispersion experiments in different regimes of radiofrequency field strengths: at low RF field strength, isotropic-chemical-shift fluctuation leads to "Bloch-McConnell-type" relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased R 1ρ rate constants ("Near-Rotary-Resonance Relaxation Dispersion", NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemicalshift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.
Journal of Biomolecular NMR, 2013
A comprehensive analysis of the dynamics of the SH3 domain of chicken alpha-spectrin is presented, based upon 15 N T 1 and on-and off-resonance T 1q relaxation times obtained on deuterated samples with a partial back-exchange of labile protons under a variety of the experimental conditions, taking explicitly into account the dipolar order parameters calculated from 15 N-1 H dipoledipole couplings. It is demonstrated that such a multi-frequency approach enables access to motional correlation times spanning about 6 orders of magnitude. We asses the validity of different motional models based upon orientation autocorrelation functions with a different number of motional components. We find that for many residues a ''two components'' model is not sufficient for a good description of the data and more complicated fitting models must be considered. We show that slow motions with correlation times on the order of 1-10 ls can be determined reliably in spite of rather low apparent amplitudes (below 1 %), and demonstrate that the distribution of the protein backbone mobility along the time scale axis is pronouncedly non-uniform and non-monotonic: two domains of fast (s \ 10-10 s) and intermediate (10-9 s \ s \ 10-7 s) motions are separated by a gap of one order of magnitude in time with almost no motions. For slower motions (s [ 10-6 s) we observe a sharp *1 order of magnitude decrease of the apparent motional amplitudes. Such a distribution obviously reflects different nature of backbone motions on different time scales, where the slow end may be attributed to weakly populated ''excited states.'' Surprisingly, our data reveal no clearly evident correlations between secondary structure of the protein and motional parameters. We also could not notice any unambiguous correlations between motions in different time scales along the protein backbone emphasizing the importance of the inter-residue interactions and the cooperative nature of protein dynamics.