Related Experiment Video
Updated: Jul 14, 2026
11:10
Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves
Published on: March 9, 2014
Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles
1Department of Chemistry, Cornell University, Ithaca, New York 14853.
Biochemistry
|October 23, 1990
Summary
Researchers mapped specific sites on chloroplast coupling factor using fluorescence methods. The N,N'-dicyclohexylcarbodiimide-binding site is ~8.5 A below the membrane surface, and the gamma-disulfide site is ~16 A above.
Area of Science:
- Biochemistry
- Membrane Biophysics
- Protein Structure
Background:
- Chloroplast coupling factor (CF) plays a crucial role in ATP synthesis.
- Understanding the spatial arrangement of CF within the membrane is vital for elucidating its function.
- Previous studies have provided limited information on the precise location of specific CF sites relative to the membrane bilayer.
Purpose of the Study:
- To determine the spatial relationship of specific sites on chloroplast coupling factor to the bilayer surface.
- To map the distances of the N,N -dicyclohexylcarbodiimide-binding site and the gamma-polypeptide disulfide site to the membrane center.
- To estimate the perpendicular distances of these sites from the membrane surface.
Main Methods:
- Fluorescence resonance energy transfer (FRET) measurements were employed to determine distances.
- Specific sites on chloroplast coupling factor were labeled with fluorescent probes (N-cyclohexyl-N -pyrenylcarbodiimide and a coumarinyl derivative).
- The bilayer center was labeled with a cholesterol derivative; fluorescence quenching by doxylstearic acids was used to determine site depth.
Main Results:
- FRET measurements indicated distances of 15 A and 43 A from the bilayer center for the N,N -dicyclohexylcarbodiimide-binding site and the gamma-disulfide site, respectively.
- A model was developed suggesting the N,N -dicyclohexylcarbodiimide site is 6-10 A below the membrane surface.
- The gamma-disulfide site was estimated to be approximately 16 A above the membrane surface.
Conclusions:
- The study provides a structural model for the spatial arrangement of key sites on chloroplast coupling factor within the membrane.
- The findings offer insights into how chloroplast coupling factor interacts with and is positioned relative to the lipid bilayer.
- This model serves as a foundation for future, more detailed structural investigations of chloroplast coupling factor.
More Related Videos
Related Concept Videos
01:24Protein Transport to the Stroma
Chloroplasts are triple membrane structures with an outer membrane, an inner membrane, and a thylakoid membrane, each containing distinct metabolite transporters, membrane translocons, and enzymes. Appropriate sorting and translocating these proteins to their correct membrane systems is essential for chloroplast function.
Protein complexes called the translocon of the outer chloroplast membrane or TOC complex, and the translocon of the inner chloroplast membrane or TIC complex mediate the...
Protein complexes called the translocon of the outer chloroplast membrane or TOC complex, and the translocon of the inner chloroplast membrane or TIC complex mediate the...
01:11Protein Transport to the Outer Chloroplast Membrane
Chloroplast outer membrane proteins encoded by the nucleus are synthesized in the cytosol. Soon after synthesis, they bind cytosolic factors such as 14-3-3 protein and the Hsp70 chaperones that keep these precursors in an unfolded state until their translocation.
Two models describe the mechanism of precursor recognition and entry across the outer membrane through the TOC complex. Model 1 suggests the newly synthesized precursor binds to the TOC receptor 159 and forms a complex.
Two models describe the mechanism of precursor recognition and entry across the outer membrane through the TOC complex. Model 1 suggests the newly synthesized precursor binds to the TOC receptor 159 and forms a complex.
01:19Membrane Asymmetry Regulating Transporters
Enzymes like flippase, floppase, and scramblase transfer phospholipids from one layer to another in the membrane, thereby affecting membrane asymmetry.
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...
01:28Protein Translocation Machinery on the ER Membrane
The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
01:22Protein Transport to the Thylakoids
Thylakoids are membrane-bound sac-like structures within the chloroplast that serve as sites for photosynthesis. Thylakoid lumen contains many electron transport proteins and is enclosed by a thylakoid membrane rich in the light-harvesting complex. Proteins targeted to the thylakoids are transported as precursors and are sorted by the general TOC/TIC import pathway. Once the precursor reaches the stroma, stromal processing peptidases remove their transit signal and expose thylakoid signal...
01:18Protein Transport to the Inner Chloroplast Membrane
Proteins targeted to the inner chloroplast membrane, or plastid proteins, are transported by two general pathways: the stop-transfer and the re-insertion or post-import pathways. Most plastid proteins carry N-terminal transit sequences and internal import sequences targeting it to the specific chloroplast subcompartment. Proteins targeted by the stop-transfer pathway have internal hydrophobic sequences that inhibit their translocation into the stroma. As a result, these precursors are arrested...