Membrane proteins account for 1/4-1/3 of total 30000 proteins that human genome encoded. Membrane proteins play an essential role in various complicated and unique cellular processes including materials transportation, cell recognition, immune response, signal transduction and regulation, and energy transfer, et.al. Almost 70% of the known or investigational drug targets are membrane proteins. It is still a remaining challenge to determine the structures and perform functional assays of membrane proteins.
Creative Biostructure has established an excellent membrane protein gene-to-structure service platform by a group of experienced professionals. Our full-set membrane protein services including expression & purification, crystallization & determination, and various functional analysis both in vivo and in vitro, proceeding your scientific research at an accelerating and exiting pace. Creative Biostructure can design and provide custom Mempro™ Fluorescence Resonance Energy Transfer (FRET) analysis or FRET assay for function research of membrane protein interactions.
Protein-protein interactions are crucial to signaling networks of membrane proteins. However, Fluorescence resonance energy transfer can only take place if the donor-acceptor distance is no more than 10 nm, making it a very powerful tool to detect and determine membrane protein interactions .
Fluorescence resonance energy transfer (FRET) assay, one of our most advanced and desirable method with extensive application range, performs assays to directly detect the oligomerization state and oligomerization degree of membrane proteins in their native environment. FRET is an distance-depended interaction between the fluorescent donor-acceptor pairs in close proximity, in which fluorescence energy is transferred from an excited donor to a suitable acceptor molecule non-radiatively. The efficiency of FRET extremely depends on the donor-acceptor distance and on overlap spectra of donor-emission and acceptor-excitation.
Figure 1. Schematic graph of a photophysical process-FRET (Molecules, 2012)
FRET can only take place if the donor-acceptor distance is no more than 10 nm, making it a very powerful tool to detect and determine membrane protein interactions. Creative Biostructure can provide Mempro™ FRET platform to perform custom membrane protein structural and functional analysis.
Table 1. Popular FRET donor-acceptor pairs and their relevant photophysical properties.
Optimal Conditions for FRET:
1. Donor-acceptor pair must be in close distance (typically 1–10 nm).
2. Overlap between acceptor absorption spectrum and donor emission spectrum.
3. Orientations between donor and acceptor must be approximately parallel.
Figure 2. Intramolecular and intermolecular FRET (Current Opinion in Structural Biology, 2001)
Figure 3. Application of sigle-molecule FRET (J. Am. Chem. Soc., 2013)
Figure 4. Measuring interaction between membrane proteins and both lipids and ligands by FRET (PNAS, 2013)
Creative Biostructure also provide an array of Mempro™ functional assays services. Please feel free to contact us for a detailed quote.
H. C. Ishikawa-Ankerhold, et al. (2012). Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM. Molecules, 17(3): 4047-4132.
K. Truong and M. Ikura. (2001). The use of FRET imaging microscopy to detect protein–protein interactions and protein conformational changes in vivo. Current Opinion in Structural Biology, 11: 573-578.
W. Bae, et al. (2013). Real-Time Observation of Multiple-Protein Complex Formation with Single-Molecule FRET. J. Am. Chem. Soc., 135(28): 10254-10257.
C. Matsushita, et al. (2013). Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides. Proc. Natl. Acad. Sci. USA, 110(5): 1646–1651.