Membrane Proteins Flashcards
Non-Specific Protein-Lipid Interactions
POST TRANSLATIONAL LIPID ADDITION
* The addition of lipids predominantly to the N- and C-termini and cysteine residues can promote association
* Can be stable or dynamic depending on the linkage
CHARGE ASSOCIATION
* Interactions with either charged head groups, hydrophobic lipids, or both, can mediate membrane-association
* For example amphipathic helicies
Specific Protein-Lipid interactions
- Specific phospholipid binding domains are common in membrane-associated signalling systems
- Ions can assist stability, or regulation of lipid binding
- Can specifically recognise head groups for targeting
Hydropathy Plot
A plot of how hydrophobic a stretch of a protein sequence is.
* transmembrane helicies will always have positive hydropathy (more hydrophobic)
* However, not very useful for β barrels
Screening for Detergents
A major hurdle for researching membrane proteins
Need to get the protein out of the membrane and keep it happy
* But membrane proteins can be fussy with what lipids are around and therefore what detergent is around — and detergent is $$$ so you need to screen
Hydrophobic tail and hydrophillic head of detergent form a micelle. The detergent interacts with hydrophobic regions and replace the lipid molecules
Eventually isolate within detergent
Intracellular GCPR pathway
- Activation of GPCR
* Ligand agonist bonds to recepter
* Active site causes conformational change which binds the heterotrimeric complex.
* Causes a conformational change that releases GDP and binds GTP.
* The heterotrimeric complex completely dissociates to trigger distant pathways
* λβ and α subunit splits - α subunit activates phospholipidase C (PLC)
* Phospholidase C is an enzyme that cleaves PIP2 into DAG and IP3 - DAG and IP3
* DAG stays associated with the membrane
* IP3 then binds to molecules in the endoplasmic reticulum which activates the opening of calcium channels which release calcium intracellularly - Activation of Protein Kinase C
* The increase in intracellular calcium helps to activate PKC with the help of DAG
* Protein kinase C turns substrate into product - Regeneration of heterotrimeric G-protein
* The α subunity completes a GTP –> GDP clevage and the G-protein begins to reassemble
Structure and conformational change of GPCRs
- 7 transmembrane helicies
- intracellular loops – loop 3-4 and 5-6 are important for mediate interaction with the α subunit
- C-terminal tail lipidation - important for interacting with λβ subunits
- N-terminal Glycosylation cab affect folding and extracellular trafficing
conformational change in the binding site
* Inactive site – helix 6 and 3 are close together and interfacing to blocking binding site
* Active site - helicies 6 and 3 move apart allowing helicies 5 and 7 to move inward. This forms a cleft which is the α binding site. The λβ subunits bind on the c-terminal lipid
GPCR families
Class A
Rhodopsin family
* small molecule ligands
* the largest class
GPCR families
Class B
Secretin family
* polypeptide hormones
GPCR families
Class C
Glutamate family
* orthosteric binding site
* additional domain (PACMAN!)
GPCR families
Class F
Frizzled
* have additional domains
* large binding partners
Type 1 integral membrane protein
Extracellular N-terminal
Intracellular C- Terminal
Type 2 integral membrane protein
Extracellular C-term
Intracellular N-term
Type 3 integral membrane protein
Multipase membrane protein
Myristolyation
- C14 saturated chain derived from myristic acid
- requires:
- N-terminal lysine residue and a stable bond to an amide
- thioester linkage
- Attached in the cytoplasm, can mediate membrane association, protein-protein interaction
Kinda reversible
Palmitoylation
- C16 saturated chain attached to a cysteine residue
- Almost excusively on the cytoplasmic face of the plasma membrane
- Readily removed by palitoyl thioesterases
- regulated membrane association
- DYNAMIC stability, reversible
Prenylation
- Thioester linkage to cysteine residue
- Recognize CaaX box near C-terminus of protein
- Mainly intracellular membranes and intraceullalar face of the plasma membrane
Protein Kinase C domains
C2 domain - Calcium is key
* Eight stranded β sandwich
* The loops of the β sandwich bind to Ca2++
* Ca2++ dependent binding to lipids
C1 domain - bound by DAG
* Small cystein rich domain
* hydrophobic residues bind and recognize DAG
* Zinc stabilizes
work together to activate the catalytic domain which phosphorylates substrate
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