Measuring transverse relaxation in highly paramagnetic systems

Abstract

The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50–400 s−1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues.

Molecular Structure of Cu(II)-Bound Amyloid-β Monomer Implicated in Inhibition of Peptide Self-Assembly in Alzheimer’s Disease

“…Paramagnetic relaxation enhancement (PRE) provides structural insights into metalloproteins that contain a paramagnetic metal ion. − Several factors may contribute to paramagnetic relaxation; for a non-blue Cu­(II) chromophore like the present case, the observable PRE is fully due to magnetic dipolar interactions between the nucleus and the paramagnetic metal ion. This allows to correlate the measured PRE value with r –6 , where r is the distance between the nucleus and the paramagnetic center. − …”

Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

“…At least 20 peaks are undetected, and these most likely correspond to the residues that fall in the "blind sphere", the region that surrounds the paramagnetic 2Fe-2S cluster [42]. Even with the use of paramagnetic-tailored experiments it was not possible to detect all the expected resonances [43,44]. Nonetheless, upon the addition of ferrichrome to FhuF, spectral changes were observed (Fig.…”

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

“…To date, the most widely used experiment [94,95] provides very accurate R 2 rates for R 2 ≤ 50 s −1 , however this approach loose accuracy in the range 50-90 s −1 and it is not applicable for R 2 > 100 s −1 . In the 1 H R 2 -weighted 15 N-HSQC-AP experiment [96], shown in Figure 4B, the relaxation delay is embedded within the INEPT evolution, the refocusing INEPT is removed and signal is acquired as an antiphase doublet as soon as the H y N z magnetization is created by the last 1 H 90 • pulse. In this very simple sequence, 1 H transverse relaxation is active only during the delay T, when the 2H x N z coherence evolves from H y as H x N z sin (πJ HN T).…”