Mechanism of long-chain fatty acid transport through fadl

The National Institute of General Medical Sciences awarded the University Enterprises Corporation at California State University, San Bernardino $183,750 on August 15, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to fund research on the mechanisms of long-chain fatty acid transport through the FADL protein in Escherichia coli.

The research addresses knowledge gaps in how the FADL transporter, an outer membrane protein in gram-negative bacteria, recognizes and translocates long-chain fatty acids and other hydrophobic substrates. The work employs substituted cysteine accessibility method and disulfide crosslinking to analyze plug domain dynamics and determine whether conformational rearrangement is required for transport. Site-directed chemical crosslinking with bifunctional crosslinkers will test whether accessibility of the lateral opening in the barrel wall is functionally linked to fatty acid translocation. The research also aims to identify specific amino acid side chains critical for substrate binding.

The award covers the period from August 15, 2026, through April 30, 2030, with performance at Verdemont, California. The assistance type is Project Grant.

Abstract

To date, transporters in the fadl family are the only known outer membrane proteins in gram-negative bacteria involved in the uptake of hydrophobic molecules, including fatty acids, alkanes, aromatic hydrocarbons, and other xenobiotics. As the paradigm of this family, fadl, the long-chain fatty acid (lcfa) transporter of escherichia coli, features a 14-stranded β-barrel that is occluded on the periplasmic side by an n-terminal plug domain. Previous studies support a lateral diffusion model, in which lcfas traverse laterally through the barrel and diffuse into the inner leaflet of the outer membrane via a conserved lateral opening in the barrel wall, while the periplasmic end remains sealed throughout the process. Despite this model, the role of the plug domain in lcfa transport remains poorly understood. IT is unclear whether the plug domain undergoes conformational changes during transport that might permit direct passage of fatty acids into the periplasm, consistent with a classical diffusion mechanism. Likewise, the molecular basis of substrate recognition by fadl is not fully elucidated. In particular, the specific amino acid side chains critical for lcfa binding have yet to be identified. The overall objective of this research is to address these knowledge gaps by elucidating the mechanisms underlying substrate translocation and molecular recognition in fadl. First, we will use the substituted cysteine accessibility method (scam) and disulfide crosslinking to analyze the dynamics of the plug domain and determine whether a conformational rearrangement of the plug domain is required for transport. Second, site-directed chemical crosslinking with a range of bifunctional crosslinkers will be employed to manipulate the permeability of the lateral opening. Coupled with transport assays, this will test whether the accessibility of the lateral opening is functionally linked to lcfa translocation. Finally, alanine and arginine scanning mutagenesis will be applied to the putative lcfa-binding sites in fadl. Binding affinities of the resulting mutants will be measured to identify key amino acid residues involved in substrate recognition. Achieving these research goals will significantly advance our understanding of the fadl transport mechanism and provide molecular-level insights to support the development of novel therapeutics targeting gram-negative pathogens, as well as strategies for the environmental remediation of pathogenic and carcinogenic xenobiotics. The significance of this research project also lies in its strong contribution to enhancing the biological research capacity and student-science engagement at california state university, SAN bernardino (csusb). This project will propel the ongoing work in the PI's laboratory to the next level while providing a unique opportunity for undergraduate students to engage in hands-on innovative research. Through this project, students will receive systematic and rigorous scientific training, establishing a strong foundation for future academic and professional development.