NIH grant: Mechanism of Long-Chain Fatty Acid Transport Through FadL (1R16GM165987-01)

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NIH grant 1R16GM165987-01: Mechanism of Long-Chain Fatty Acid Transport Through FadL
Institute: {'code': 'GM', 'abbreviation': 'NIGMS', 'name': 'National Institute of General Medical Sciences', 'admin_org_id': '12500024', 'admin_funding_url': ''}. PI: Xiaoxu  Jiang. Award: $183750.
Start: 2026-08-15T00:00:00.
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.

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