GIST Repository
College of Life Sciences and Medical Engineering
Department of Life Sciences
3. Theses(Master)
Cryo-Electron Microscopy Study of the human Citrate Transporter NaCT
- Abstract
- NaCT, a membrane transporter within the SLC13 family, plays a critical role in cellular metabolism by mediating sodium-dependent citrate transport. In this study, I conducted a comprehensive analysis of human NaCT (hNaCT) through structural, functional, and inhibitory assays. The purified hNaCT protein was reconstituted into nanodiscs for structural studies, revealing its inward-facing conformation at a resolution of 2.75 Å via Cryo-electron microscopy (Cryo-EM). Functional assays highlighted the critical influence of sodium and pH gradients on citrate transport, with maximum activity observed at pH 6.0 and an external sodium concentration of 200 mM. nano-Differential Scanning Fluorimetry (nanoDSF) experiments demonstrated that SLC13A5-IN-1 provided the highest thermal stabilization for hNaCT at pH 7.5. However, transport assays identified PF-06649298 as the most potent inhibitor of hNaCT activity, followed by PF- 06761281, BI01383298, and SLC13A5-IN-1. The discrepancy between thermal stability and inhibitory efficacy may arise from differences in solubility and binding mechanisms. These findings offer novel insights into the structure and function of hNaCT, providing a foundation for the development of targeted therapeutics for metabolic disorders. Further studies are needed to refine inhibitor binding models and evaluate their clinical potential.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Kim Narim
- Department
- 대학원 생명과학부
- Advisor
- Jin, Mi Sun
- Table Of Contents
- ABSTRACT ⅰ
LIST OF CONTENTS ⅱ
LIST OF TABLES AND FIGURES ⅳ
I. INTRODUCTION 1
II. METHOD
2.1. hNaCT Cloning and expression 4
2.2. hNaCT purification 4
2.3. Nanodisc reconstruction 5
2.4. nano-Differential Scanning Fluorimetry(nanoDSF) assay 6
2.5. Proteoliposome reconstitution 6
2.6. Citrate Transport assay 7
2.7. hNaCT Cryo-EM grid preparation, data collection and processing 7
III. RESULTS
3.1. Expression Analysis of hNaCT and hNaCT Antibodies 9
3.2. Detergent screening and Purification of hNaCT 9
3.3. Purification of hNaCT Antibodies 9
3.4. hNaCT Nanodisc Preparation and Fv Binding Screening 10
3.5. Functional and Inhibitory Analysis of hNaCT Transport 10
3.6. Inward State of hNaCT Cryo-EM Data 11
IV. DISCUSSION 12
V. FURTHER STUDY 13
VI. REFERENCES 32
VII. ACKNOWLEDGEMENT 34
LIST OF TABLES AND FIGURES
Table 1. Protein construct list 14
Table 2. hNaCT grid preparation condition 15
Table 3. hNaCT data collection and processing 16
Figure 1. hNaCT gene design and expression test 17
Figure 2. hNaCT detergent screening 18
Figure 3. hNaCT purification 19
Figure 4. hNaCT-Fv purification 20
Figure 5. hNaCT-Fab purification 22
Figure 6. hNaCT Nanodisc Formation and Fv Interaction Analysis 23
Figure 7. Thermal Stability of hNaCT via nanoDSF Analysis 24
Figure 8. hNaCT Citrate Transport assay 26
Figure 9. hNaCT Cryo-EM grid screening result 28
Figure 10. hNaCT Cryo-EM process workflow in nanodisc 29
Figure 11. The Cryo-EM data processing results of hNaCT in nanodisc 30
- Degree
- Master
- Appears in Collections:
- Department of Life Sciences > 3. Theses(Master)
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