Triple Quantum Decoherence under Multiple Refocusing: Slow Correlated Chemical Shift Modulations of C′ and N Nuclei in Proteins

Abstract

A new experiment allows the identification of residues that feature slow conformational exchange in macromolecules. Rotations about dihedral angles that are slower than the global correlation time tau(c) cause a modulation of the isotropic chemical shifts of the nuclei. If these fluctuations are correlated they induce a differential line broadening between three-spin single-quantum and triple-quantum coherences involving three nuclei such as the carbonyl C', the neighbouring amide nitrogen N and the amide proton H(N) belonging to a pair of consecutive amino acids. A cross-correlated relaxation rate R (CS/CS)(C'N) can be determined that corresponds to the sum of the isotropic and anisotropic contributions to the chemical shift modulations of the carbonyl carbon and nitrogen nuclei. Only the isotropic contributions depend on the pulse repetition rate of a multiple-refocusing sequence. An attenuation of the relaxation rate with increasing pulse repetition rate can therefore be attributed to slow motions. The asparagine N25 residue of ubiquitin, located in the first alpha-helix, is shown to feature significant slow conformational exchange.

Slow motions in nondeuterated proteins: Concerted chemical shift modulations of backbone nuclei

“…In spite of the strong dipolar contributions in the nondeuterated sample, the measured rates AR(DQC/ZQC) permit the identification of residues that experience significant conformational exchange, such as asparagine 25 [10]. Figure 3d shows the very good correlation (r = 0.97) between the rates obtained by applying the sophisticated TCQ/SQC experiment to (expensive) deuterated ubiquitin and the rates obtained with the simple DQC/ZQC experiment in (cheap) nondeuterated ubiquitin.…”

“…The earlier TQC/SQC{C'NH s} method [10] was designed to measure the difference AR(TQC/SQC) in autorelaxation rates of TQC(C+N+H N) and three-spin SQC(C+N_HN). In this paper, we shall report measurements of rates AR(DQC/ZQC) which are obtained from the autorelaxation rates of DQC(C'N+) and ZQC(C+N ).…”

“…In this paper, we show that DQC/ZQC experiments involving only C' and N nuclei (instead of the TQC/SQC expe¡ described previously [10]) can be used for the identification of residues experiencing conformational exchange, in spite of interferences both from the attached H N proton and from remote protons such as H ~, provided that these dipolar contributions do not vary significantly from one residue to another.…”

“…Thus, measurements of CS-CS rates asa function of pulse repetition rates enable the separation of CSM/CSM (rank 0 interactions) from CSA/CSA (rank 2 interactions). A TQC/SQC{C'NH N} method involving a carbonyl C' nucleus, the neighboring amide N nucleus, and the attached amide proton H N, can provide evidence of slow backbone motions [10]. More recently, a DQC/ZQC{NH n} experiment involving only an amide N nucleus and its attached proton H N has been shown to provide useful information [11].…”

Using Protein Motion to Read, Write, and Erase Ubiquitin Signals

“…nanosecond) timescale, a portion of ubiquitin's core near the N terminus of ubiquitin's ␣-helix has been known to be dynamic on the much slower microsecond to millisecond timescale since the early 1990s (23)(24)(25). Further characterization of these motions with spin relaxation experiments identified that this spatially clustered region fluctuates with a timescale of ϳ40 -100 s (25)(26)(27)(28)(29). This same region has also been observed to be mobile in a recent 1-ms molecular dynamics simulation of the native state of ubiquitin (30).…”