Lecture 2: Membrane Proteins Flashcards
What types of transmembrane domains can we have?
NOTE: TM beta-strands - barrel structure
- Anchored - have regions interacting with the membrane but the other half doesn’t (could e.g. bind ligands)
Phospolipids make up the membrane
- hydrophillic heads - intermediate - hydrophobic tails
What measure can help us predict the structure of transmembrane proteins?
Higher hydropathy index -> the more hydrophobic an amino acid side chain
-> we can create hydropathy plot
- shows us regions of high lipophilic character of a protein -> good predictor of the structure of transmembrane proteins
- e.g. anchored - one side of chain has risinf HI while the other decreasing, barrel - every first facing inside while every second outside
Look at concrete examples:
What is a crutial thing that helps with integrating proteins into the membrane? How does it work>
Signal Recognition Particle (SRP) = protein that can recognize signaling peptide sequences -> binds to it
- SRP tends to be located towards the N-terminus
=> signaling sequence initiates ribosome traveling to ER -> binding of SRP stops the translation of protein -> guided to their endoplasmatic receptor (rough ER - ribosomes are bind to the surface) -> binding to Translocon which opens -> Translation can continue + SRP dissociates (ready for a new cycle)
- What happens to the protein?
- Translocon is open -> chain passes through -> once signaling sequence reaches inside of ER lumen. it gets cleaved off by Signal peptidase -> protein translation goes through into lumen
In contrast to pure ER localization - how does membrane insertion work?
- Ribosom binds to translocon -> goes through
- Signal peptidase cleved off the signal sequence
- Translation continues here
- Stop-transfer anchor sequence = sequence of aa that stop the translocon
- aa starts forming alpha helix = blocks for a while (accumulates)
- Once signal anchor sequence gets produced the whole helix is pushed out of the Translocon
- More translation is conducted withincytoplasm (before ribosome dissociates)
NOTE: Depending on location of signaling sequences we can get different lengths of C and N termini
Look at a similar case - but with N terminus outside and C-terminus inside:
What types of membrane proteins do we have?
Type 1 - relatively larger N term with short C
Type 2 - larger C and shorter C (reverse orientation)
Type 3 - similar to type 1 just with large C and smaller N
Type 4 - several spanning proteins, sophisticate interaction between signalling sequences
Why is it hard to determine structure of membrane proteins?
- They are accustomed to amphipathic environment - half/half is hard to create in a solution
-> we need to perform solubilization with detergines
- just like proteins have hydrophobic tail and hydrophilic head => can shield the hydrophobic patched of the protein with their own hydrophobic tails and hydrophilic heads shield as well
NOTE: in crystalization we need to organize them - difficult task
What other technique could be used?
Truncations and fusion:
- maybe we are interested only in the “outside” area (not transmembrane) -> fusion proteins can pull it out and shield/stabilize it in solutions
- Could also use antibodies to stabilize
NOTE: Used for crystalography
How do rods look like?
All the discs = layered membranes with high density of Rhodopsin - part of a larger signaling cascade of G-protein Coupled Receptor
In brief general terms - how do G-protein coupled receptors work?
- GPCRs 7 TM spanning (green)
- G-protein
- subunits: alpha, beta, gamma
- has two states: ON (GTP) or OFF (GDP)
-alpha: classical G, membrane anchored - beta, gamma: heterodimer, membrane anchored
Describe step by step general G-protein functioning.
- Ligand binds -> conformation change
- G receptor is activated
- G protein binds to activated receptor -> conformational change in alpha
- Kicks out GDP and is replaced by GTP (high concentration in cytosol)
- Makes Galpha leave (while beta and gamma stay)
- Ligand dissociates, Galpha activates effector protein
- Hydrolysis of GTP to GDP -> alpha dissociates and meets beta and gamma
Many different types of alpha-subunits cause different cellular effects
So how does the G-protein receptor work in retina?
- Light enters the retina
- Inactive Opsin gets activated
- G-protein comes in = Transducin -> dissociation (GDP->GTP)
- Alpha interacts with cGMP-Phospodiesterase -> starts converting cGMP into GMP
- Low concetration of cGMP close cGMP gated ion channels (normally Na+ and Ca+ influx)
What happens inside Rhodopsin?
- Has cofactor Retinal - reacts to light
-> normally 11-cis conformation -> upon light changes to all-trans retinal - Helix 5 and 6 clash with transducin in dark state
What are some characteristics of potassium channel?
= Multiple Helix Transmembrane spanning protein
- Tetrameric -> each of the subunits consists of 2 helices and a pore helix (the ones that make up the pore)
- 10000x better conductivity for K+ and Na+
How should we actually depict ions in solutions? How does K+ pass through its channel?
We also have to think about ions being “caged” in water (hydrogen shell) -> and how much energy would we need to break these hydrogen bonds
Ions enter the pre-chamber via a wide opening -> goes into selectivity filter
- consist of pore helix which stabilizes with the negative part of the helix = positions 3 carbonyl groups of the backbone creating a channel
- this channel cannot be entered by H2O
-> K+ has to be de-hydrated, which needs energy
-> attaching negative carbonyl group evens out energetically the breakadge of hydrogen water bonds
How come only K+ passes and NOT Na+ - especially considering that the latter is much smaller?
Since Na+ is smaller when it gets to the selectivity filter it cannot simultaneously reach all 4 carbonyl groups (it can only do 2)
-> so the freed energy (from binding to carbonyl groups) cannot compensate the breaking energy of hydrogen bonds
=> very unlikely to pass
What is the respiratory chain?
= orchestrated electron transport chain that reduces O2 to H2O
- while doing so it let’s electrons pass through the system -> build up positive charge/gradient of the membrane
-> proton (H+) gradient is build up -> energy from it is used to:
- drive import of ADP (via antiport of ATP)
- drives import of Pi
- drives ADP phosphorylation
How does ATP look? What is it?
= adenosine triphophate
- nucleotide composed of adenine base + ribose + 3 phosphate groups (alpha, beta, gamma - last missing in ADP)
- phosphate groups has strong negative charge, breaking the bond frees a lot of energy
How does ATP-Synthase looks like?
= Transmembrane complex with 2 subunits: F0 (membrane, ring), F1 (matrix)
- F0 = proton channel, F1 = ATPase, alpha and beta - each binding ATP and ADP + gamma connected to F0
How does ATP-Synthase works in terms of chemical reactions and mainly changes in the protein?
- Chemical mechanism
- ATP is synthesized from ADP and Pi in the beta-subunit of F1
- Protein
- synthesis is fairly easy BUT the release of ATP gets troublesome -> need for gamma subunit to engage in rotation -> changes interactions with beta -> induces conformational changes in beta -> ATP can be kicked out
- goes through cycles of OPEN (O), LOOSE (L), TIGHT (T):
- O state - ADP and Pi can binds -> becomes L conformation -> becomes T conformation which is where chemical reaction takes place, ADP and Pi are very close to one another -> O conformation = ATP leaves -> ADP and Pi comes in
=> we need gamma rotation to bring all these conformational changes
Look at picture of a ring structure (brown)
There are a lot of different C-rings possible -> humans tend to have 12 C units
What components do we need for c-ring to move?
Components:
- high concentration of H+ outside the membrane
- C-ring structure (12 subunits)
- Aspertate 61 present in each subunit
- it is possible to be in protonated form (not normally done)
- A region connecting the ring structure with the F1D
= it is a half channel, there is passage from intramembrane space to a position that is negatively charged (attracts H+) - but is just half so it doesn’t go completaly
