Paramagnetic ions (Cu²⁺, Mn²⁺, etc.) can be used as atomic rulers in Nuclear Magnetic Resonance (NMR) spectroscopy to probe long distances between these ions and various NMR active nuclei (> 1 nm). Paramagnetic ions such as Cu²⁺ (S = ½) cause a strong paramagnetic relaxation enhancement (PRE) to nearby nuclei in space (< 1 nm), which causes peaks to extremely broaden, or disappear, beyond detection. Nuclei that are further away (1-2 nm) from the Cu²⁺ paramagnetic ion experience intermediate peak broadening on the order of ~ 5 – 100 Hz. The precise extent of peak broadening, or the transverse PRE, can aid in structure elucidation and the determination of intermolecular interactions between molecules.
To demonstrate this methodology, a uniformly ¹⁵N-labeled K28H GB1 protein sample was expressed and purified as a control. Herein, it is shown that Cu²⁺ and iminodiacetic acid (IDA) can strongly bind to the mutated solvent-exposed histidine residue. 2D ¹H-¹⁵N HSQC spectra were recorded on the diamagnetic reference (no Cu²⁺) and in the presence of Cu²⁺. By measuring the peak volume ratio of the paramagnetic peaks and diamagnetic peaks, distances were extracted using the ¹H transverse PRE equation for each Cu²⁺ to backbone amide amino acid.
Since 2D ¹H-¹⁵N HSQC spectra on ¹⁵N uniformly labeled proteins can contain peak overlap and be time-consuming, this project mainly aims to measure ¹H transverse PREs using a ¹⁵N-labeled phenylalanine K28H GB1 sample. To do this, a selectively ¹⁵N-labeled phenylalanine K28H GB1 protein sample was expressed and purified. Since there are only two phenylalanine peaks (F30 and F52) in the protein that conveniently have resolved ¹H chemical shifts, a 1D ¹⁵N-filtered ¹H NMR spectrum was recorded with and without the paramagnetic ion. Since F30 is very close to the Cu²⁺/IDA binding site at the 28H position, it demonstrated a greater ¹H transverse PRE than F52. The distances extracted from the 1D spectra were compared to those extracted from the 2D spectra, which were both compared to the predicted distances from the known protein structure. Ultimately, this method proves to be useful for 1) large proteins with lots of peak overlap in 2D ¹H-¹⁵N HSQC spectra and 2) pedagogical purposes in an undergraduate spectroscopy course to demonstrate the power of PREs in protein NMR.

Jake Simmons

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