Biophysical Characterization of Human Protamine-1 as a Responsive CEST MR Contrast Agent

Abstract

The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect chromosomal DNA in germ cells by packing it densely using electrostatic interactions. Human protamine-1 (hPRM1) has been developed as a magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) reporter gene, based on a sequence that is approximately 50% arginine, which has a side chain with rapidly exchanging protons. In this study, we have synthesized hPRM1 and determined how its CEST MRI contrast varies as a function of pH, phosphorylation state, and upon noncovalent interaction with nucleic acids and heparin (as antagonist). CEST contrast was found to be highly sensitive to phosphorylation on serine residues, intra- and intermolecular disulfide bridge formation, and the binding of negatively charged nucleotides and heparin. In addition, the nucleotide binding constants (K_eq) for the protamines were determined through plotting the molar concentration of heparin versus CEST contrast and compared between hPRM1 and salmon protamine. Taken together, these findings are important for explaining the CEST contrast of existing arginine-rich probes as well as serving as a guideline for designing new genetic or synthetic probes.

Figure 1

MTR_asym of different protamine solutions at various conditions: (A) Comparison of CEST contrast of protamine sulfate and synthetic hPRM1 at the same concentration 0.733 mM in PBS (B₁ = 3.6 μT, T_sat = 4 s); (B) Comparison of CEST contrast for mono-, di-, and nonphosphorylated forms of synthetic hPRM1 (B₁ = 3.6 μT, T_sat = 4 s); (C) hPRM1 CEST contrast in the presence of negatively charged single nucleotides and polymeric heparin molecules using saturation pulse with B₁ = 3.6 μT, T_sat = 4 s; (D) pH dependence of hPRM1 CEST contrast done at B₁ = 3.6 μT, T_sat = 4 s.

Figure 2

Proposed conformation model of hPRM1 displaying the multiple cysteines involved in intramolecular disulfide bonding (yellow) and intermolecular bonding (red) based on the Vilfan et al., 2004 model for bull protamine.

Figure 3

Cartoon of CEST contrast of hPRM1 alone (upper) and complexed with 5′-ATP (lower), which resulted in a decrease of CEST contrast in MTR_asym. B₁ = 3.6 μT, T_sat = 4 s.