STRUCTURAL CHARACTERIZATION AND MOLECULAR TARGETS OF HUMAN β-DEFENSIN 18 USING FLUORESCENCE SPECTROSCOPY AND NUCLEAR MAGNETIC RESONANCE TECHNIQUES

Antimicrobial peptides (AMPs) are found in all living organisms and constitute the first line of defense against pathogens, including viruses, bacteria, fungi, and protozoa. They are components of innate immunity and can modulate innate responses in higher organisms. Defensins are AMPs divided into three structural subfamilies: α-, β-, and θ-defensins. Human β-defensins (HbDs) are cationic proteins sharing a conserved structure composed of three β-strands forming an antiparallel β-sheet stabilized by three disulfide bonds and flanked by an N-terminal α-helix. Their proposed antimicrobial mechanism involves pore formation in microbial membranes and disruption of cellular activities.

Human β-defensin 18 (HbD18), the focus of this project, is expressed in the male urogenital tract, comprises 103 residues, and has no defined structure or molecular targets. Studying β-defensins is important for understanding their interactions with pathogens and host cells and may support alternative treatments for infections amid increasing antibiotic resistance. Thus, this work aims to develop a high-yield functional heterologous expression protocol for subsequent structural characterization and interaction studies with different targets, particularly lipids mimicking the plasma membrane.

HbD18 was expressed in Escherichia coli C43(DE3) at 18 °C, resulting in soluble protein overexpression. After lysis, the protein was purified by nickel-affinity chromatography, followed by cleavage of thioredoxin and the histidine tag and a second purification by high-performance liquid chromatography (HPLC). Intrinsic tryptophan fluorescence spectroscopy was then performed with lyophilized protein resuspended in phosphate buffer. Thermal stability was evaluated in the free protein by gradually increasing the temperature from 20 to 60 °C, followed by experiments with sodium dodecyl sulfate (SDS), phosphatidic acid (PA), and CHAPS.

The results showed that HbD18 remained stable from 20 to 60 °C and exhibited fluorescence changes associated with binding to SDS and PA micelles, but no interaction with CHAPS. Future experiments will evaluate fluorescence in the presence of other surfactants and potential molecular targets, including genetic material, since defensins are present in neutrophil extracellular traps (NETs). HbD18 doubly labeled with ^15N and ^13C will also be produced for structural characterization by nuclear magnetic resonance (NMR) spectroscopy.