# Integrated approaches to symport mechanisms of membrane transporters

> **NIH NIH R01** · TEXAS TECH UNIVERSITY HEALTH SCIS CENTER · 2020 · $325,125

## Abstract

Integrated approaches to symport mechanisms of membrane transporters
PROJECT SUMMARY/ABSTRACT
 Our long-term objective is to understand the molecular mechanisms of cation/solute symport catalyzed by
membrane carriers. These transporters play critical roles in maintaining normal cellular activities, are important
in human health and disease, and can serve as drug targets and therapeutic delivery pathways. In this
proposal, we plan to study the bacterial Na+-coupled melibiose permease (MelB), which utilizes energy stored
in the electrochemical gradient of Na+, Li+, or H+ to drive the translocation of galactoside against its
concentration gradient, and is a prototype for exploring molecular mechanisms of symporters in the MFS family
that can use more than one cationic species for coupling. The MelB homologue expressed in blood-brain and
blood-retina barriers catalyzes Na+-coupled uptake of docosahexaenoic acids (DHA)-carrying
lysophosphatidylcholine (LPC), thus supplying essential DHA to brain and eyes for neural development and
prevents neurodegeneration. For secondary-active transport in general, the coupling between the driving
cation and cargo solute is obligatory, but the mechanisms underlying the energetic coupling remain largely
unknown. We will elucidate the Na+-coupled symport mechanisms by a combined approach, including
genetics, biochemistry, calorimetry, site-directed spin labeling (SDSL) with continuous-wave electron
paramagnetic resonance spectroscopy (CW-EPRs), and 3-D X-ray crystallography. We have created high-
affinity MelB-camelid single-domain nanobodies (Nbs) for crystallization of MelB, and have implemented
isothermal titration calorimetry (ITC) measurements to determine the free-energy changes and heat capacity
changes for the binding of MelB’s ligands (melibiose, Na+, Li+), alone or together, as well as the CW-EPRs to
measure ligand-induced solvent accessibility changes and proximity changes. Based on our strong preliminary
results, three independent but complementary aims are proposed to test our central hypothesis: the core of the
symport mechanism is cooperative binding of co-substrates that induces the formation of an occluded
intermediate state. Our integrated multi-disciplinary approach will provide important missing information into
the cation/solute symport mechanisms and improve our fundamental knowledge of the ligand binding
energetics and protein conformational changes in general, as well as directly impact on other studies of Na+-
coupled transporters including the LPC transporter in brain and retina.

## Key facts

- **NIH application ID:** 9969449
- **Project number:** 5R01GM122759-03
- **Recipient organization:** TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
- **Principal Investigator:** Lan Guan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $325,125
- **Award type:** 5
- **Project period:** 2018-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9969449

## Citation

> US National Institutes of Health, RePORTER application 9969449, Integrated approaches to symport mechanisms of membrane transporters (5R01GM122759-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9969449. Licensed CC0.

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