Toward Structural Dynamics: Protein Motions Viewed by Chemical Shift Modulations and Direct Detection of C′N Multiple-Quantum Relaxation

Abstract

Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein–Protein Interactions of Metalloproteins: The Case of Iron–Sulfur Proteins

“…This has been first applied in copper proteins [101,102] and in Ln(III)-substituted Calcium binding proteins [29,103] where 13 C detection significantly reduced the blind sphere due to paramagnetism around the metal ion. It has also been shown that 13 C protonless experiments significantly improve the detectability of effects such as residual dipolar couplings (RDC hereafter) involving fast relaxing 1 H spins [104] as well as multiple quantum relaxation rates [105]. For iron-sulfur proteins, Markley and coworkers pioneered the idea and extensively used not only 13 C, but also 15 N and 2 H direct detection, to obtain hyperfine shifts of heteronuclei when the corresponding 1 H signals were broadened beyond detection for rubredoxins and also for Rieske proteins [52,53,100,106].…”

Full relaxation matrix analysis of apparent cross-correlated relaxation rates in four-spin systems

“…Two values have a relatively large uncertainty in the above assumptions that must be checked: First, J I1I2 = J S1S2 is usually not strictly correct; second, (R DQ À R ZQ ) may vary substantially if exchange mechanisms are active [20,39]. As mentioned above, W 0 only contributes to ZQ but not to DQ coherence if J I1I2 = J S1S2 .…”

Distance‐independent Cross‐correlated Relaxation and Isotropic Chemical Shift Modulation in Protein Dynamics Studies

“…Cross‐correlated chemical shift modulation (CSM/CSM) is a manifestation of correlation between the local electronic environments of two spins ,,. The zero‐ and double‐quantum coherences between the two spins relax at different rates partially due to correlated modulation of the isotropic chemical shifts.…”