Direct measurement of dipole–dipole/CSA cross-correlated relaxation by a constant-time experiment

Abstract

Relaxation rates in NMR are usually measured by intensity modulation as a function of a relaxation delay during which the relaxation mechanism of interest is effective. Other mechanisms are often suppressed during the relaxation delay by pulse sequences which eliminate their effects, or cancel their effects when two data sets with appropriate combinations of relaxation rate effects are added. Cross-correlated relaxation (CCR) involving dipole-dipole and CSA interactions differ from autocorrelated relaxation (ACR) in that the signs of contributions can be changed by inverting the state of one spin involved in the dipole-dipole interaction. This property has been exploited previously using CPMG sequences to refocus CCR while ACR evolves. Here we report a new pulse scheme that instead eliminates intensity modulation by ACR and thus allows direct measurement of CCR. The sequence uses a constant time relaxation period for which the contribution of ACR does not change. An inversion pulse is applied at various points in the sequence to effect a decay that depends on CCR only. A 2-D experiment is also described in which chemical shift evolution in the indirect dimension can share the same constant period. This improves sensitivity by avoiding the addition of a separate indirect dimension acquisition time. We illustrate the measurement of residue specific CCR rates on the non-myristoylated yeast ARF1 protein and compare the results to those obtained following the conventional method of measuring the decay rates of the slow and fast-relaxing 15 N doublets. The performances of the two methods are also quantitatively evaluated by simulation. The analysis shows that the shared constant-time CCR (SCT-CCR) method significantly improves sensitivity.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

“…There are several NMR methods available for measurement of correlation times. We have used a method based on interference between 15 N relaxation contributions from chemical shift anisotropy and dipole-dipole mechanisms (37). This has an advantage over T 1 /T 2 measurements in that it is less sensitive to chemical exchange effects.…”

“…The backbone rotational correlation times were extracted for each of the assigned 15 N backbone sites using shared constanttime cross-correlated relaxation experiments (SCT-CCR) (37). These experiments allow a direct measure of rotational correlation times for N-H bond vectors, relatively independent of effects from remote protons and chemical exchange.…”

“…Once NMR resonance assignments were made on non-glycosylated material and transferred to the glycosylated material, 1 H-15 N HSQC spectra provided a structure-sensitive fingerprint that allowed comparison of the domain structure in the various constructs. They also provided a basis for measurement of rotational correlation times that are sensitive to dimer formation (37), and for the collection of residual dipolar coupling (RDC) data that are orientation dependent. The latter can more rigorously confirm the retention of the domain structure, as well as distinguish which of the dimer structures seen in crystal structures may be present in solution (38,39).…”

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors

“…To obtain τ c , 15 N NMR transverse cross-correlated relaxation was measured (Liu and Prestegard, 2008) and compared against spectral density expressions available (Lee et al, 2006). …”

Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12

“…Distance restraints were obtained from the PREs ( Γ 2 ) using an equation of ref 59 simplified by ω H τ c >> 1 to the form: $$r=4K{\tau }_{\mathrm{normalC}}∕{\Gamma }_2$$ where K is 1.23·10 −44 m 6 s −2 . The rotational correlation time τ c of protein-bicelle complexes was obtained from amide cross-correlation rates η xy 60 interpreted by the TRACT approach 61 .…”