Monitoring Binding of Protamine with DNA Using Protein Charge Transfer Spectra

• Published in: The journal of physical chemistry. B 2024-10, Vol.128 (40), p.9656-9668
• Main Authors: Priyadarshi, Anurag, Saikia, Simangka Bor, Swaminathan, Rajaram
• Format: Article
• Language: English
• Subjects: B: Biophysical and Biochemical Systems and Processes
• ISSN: 1520-6106; 1520-5207; 1520-5207
• DOI: 10.1021/acs.jpcb.4c03665

In this work, novel intrinsic electronic absorption (250–400 nm) with a molar extinction coefficient of 752 M–1cm–1 at 250 nm, arising from photoinduced electron transfer involving charged amino acid side chains and the polypeptide backbone, along with luminescence (300–500 nm) with quantum yield of 0.011 from subsequent charge recombination, was observed in salmon sperm Protamine (PRM). The absorption of PRM was attributed to the previously identified Peptide Backbone-to-Side chain Charge Transfer (PBS-CT) from the polypeptide backbone to the abundant cationic headgroups of Arginine in PRM, while the luminescence was believed to originate from charge recombination within the charge-separated excited states of PRM. Remarkably, since Arg is the only charged residue in the PRM sequence, the PRM Protein Charge Transfer Spectra (ProCharTS) is both totally and uniquely Arg specific. Interestingly, the peak of PRM luminescence emission spectrum was independent of the excitation wavelength, unlike other proteins such as human serum albumin, displaying unconventional luminescence. Aggregation-induced effects on PRM absorbance and luminescence were ruled out, as both PRM absorbance and luminescence increase maintained linearity with increasing concentration in the 25–150 μM range. Nucleoprotamine complex formation, resulting from the binding of PRM with calf-thymus genomic DNA (gDNA), was monitored through increased scattering by the nucleoprotamine complex, a decrease in gDNA/PRM absorbance, a decrease in gDNA/PRM ellipticity, and shifts of nucleoprotamine complex band upon agarose gel electrophoresis. Upon binding with gDNA (700 μM base pair concentration), PRM ProCharTS absorbance at 260 nm decreased by 72%. This decrease was attributed to the formation and subsequent precipitation of nucleoprotamine complex upon PRM-gDNA binding. The application of ProCharTS absorbance to indirectly monitor DNA–protein binding in a label-free approach was thus demonstrated.