Measuring transverse relaxation in highly paramagnetic systems

Michele Invernici,Francesca Cantini,Inês B Trindade,Ricardo O Louro,Mario Piccioli

doi:10.1007/s10858-020-00334-w

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.

Highlights

The hyperfine interaction between electron and nuclear spins gives rise to additional contributions to chemical shifts and nuclear relaxation, both of which can be used as a source of structural restraints (Piccioli and Turano 2015; Turner et al 1998)
We have shown that structural restraints derived from Paramagnetic Relaxation Enhancements (PRE) can be used as the unique source of restraints for the structure calculation of small metalloproteins (Trindade et al 2020b)
A key point for a successful PRE-only approach is the availability of many accurate relaxation rate values measured throughout the entire protein, including the close proximity of the paramagnetic center, where nuclear spins are strongly affected by paramagnetism

Summary

Introduction

The hyperfine interaction between electron and nuclear spins gives rise to additional contributions to chemical shifts and nuclear relaxation, both of which can be used as a source of structural restraints (Piccioli and Turano 2015; Turner et al 1998). Paramagnetic relaxation depends on γ2 of the observed nucleus, 13C or 15N direct detection (Arnesano 2003; Kolczak et al 1999; Lin et al 2003) have been successful as an efficient alternative to 1H detected experiments, for assignment purposes (Bermel 2005; Machonkin et al 2002; Bertini et al 2005b) and for the obtainment of PREs. the 15 N HSQC experiment still remains the “easiest” molecular fingerprint, methods to exploit the use of 1HN T1 and T2 rates are welcome

Methods

Results

Conclusion