Question

13. A structural biologist from a renowned medical research institute has just solved the structure of a protein complex formed between two proteins A and B. She wants to find out if this complex forms in living cells. She has managed to label protein A with a green fluorescent protein and protein B with a red fluorescent protein. The green and red fluorescent proteins form a Forster resonance energy transfer pair.

Explain how you might detect the presence of the A;B complex in living cells paying special attention to which technique you choose, how it works and what you would expect to see. (1 page limit)

Answer 1

Answer:

• A group of twenty different amino acids forms a protein and hundreds and thousands of these amino acids are attached together to form long chains of proteins molecules.
• Mostly proteins moves through the endoplasmic reticulum system and are dispatched from the trans-face of the golgi apparatus in transport vesicles which inturn moves through the cytoplasm and fuse with the plasma membrane releasing the protein outside the cell.
• Proteins serve a variety of functions within cells. Some are involved in structural support and movement, others in enzymatic activity.
• A cell to cell systematic movement method can be used to analyze the study where, A 35-kDa MP encoded by RNA2 is essential for the cell-to-cell and systemic movement of RCNMV.
• This RCNMV MP has the ability to bind single-stranded nucleic acids cooperatively, localizes in the cell wall and increases the size exclusion limit of the plasmodesmata.
• RCNMV MP fused with GFP (RCNMV MP:GFP) can form a homopolymer and is targeted to the cell wall, and this targeting is required for viral cell-to-cell movement.
• Retroviruses and other immunotropic viruses often utilize immunological synapses for cell-to-cell spread.
• Contact between antigen-presenting cells and T cells is initiated by an interaction between intercellular adhesion molecules (ICAMs) expressed on APCs and LFA-1, the CD11α/CD18 αL/β2 integrin.
• Following the initial contact, major histocompatibility complex (MHC) molecules carrying antigenic peptides and the T-cell receptor move into the center of the contact zone, while the integrin forms an outer ring.
• Talin binding to the cytoplasmic tail of integrins marks the outer rings. A center-directed flow driven by actomyosin lies behind these reorganizations.
• The central and peripheral zones are called the central and peripheral supramolecular activation clusters, cSMAC and pSMAC.
• Cell-cell adhesion positions the PAR complex at the cytoplasmic face of the cell-cell contact zone to establish cellular polarity.
• These events have been described in great detail for the T-cell side, polarization is often observed on both sides of the synapse.
• The concentration of both receptors in the cell-cell interface reduces the responsiveness of T cells to soluble chemokines, thereby preventing their migration and stabilizing the immunological synapse.

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