Midterm No. 2, Opus 1 Flashcards
What is membrane fluidity dependent on?
Temperature
Membranes at low temp
Gel like consistency
Membranes at high temp
Fluid like consistency
Lipids are amphipathic/amphiphilic. What does this mean?
Means lipids are hydrophobic and will self-assemble and then self-seal in aqueous environments.
What lipid shape forms micelles?
Single tail, cone
What lipid shape forms bilayers?
Double tail, cylinder
Why do lipids within a bilayer leaflet have lateral mobility?
Because their tails aren’t covalently bonded
At what speed to lateral shifts occur among lipids in a bilayer?
10^ -6 seconds
Happens all the time
At what speed to transverse shifts occur among lipids in a bilayer?
10^5 seconds
Hardly ever spontaneous
Phosphoglycerides and sphingolipids are subcategories of…
…phospholipids
Phosphoglycerides
Dominant type of lipid in membranes
Made up of a glycerol, two fatty acid tails, a phosphate group, and a polar head group
Sphingolipids
Typically have longer fatty acid chains
3 carbon linker has an amino group
They do NOT use glycerol
Very common type of lipid in membrane rafts
Important to neural tissue
Sphingolipids typically have longer fatty acid chains. Because of this…
Membranes are thicker where sphingolipids are present
Membrane sections containing sphingolipids attract different types of proteins
They are better at providing electrical insulation than lipids of normal length
How long are normal fatty acid tails in a bilayer?
Usually 3.5 nm long
The whole lipid molecule in a bilayer, including its polar head group, is normally 3.7 nm
Explain the importance of sphingomyelin to neural tissue
Oligodendrocytes with membranes enriched in sphingomyelin provide electrical insulation for axons
Sphingomyelin makes up the myelin sheath found in nerve cells
Membrane raft domains
Type of substructure within the membrane, less fluid regions within a plasma membrane
Can move laterally in the rest of the membrane lipid sea
Some viruses like to enter cells via these rafts
Rafts are often rich in sphingolipids and cholesterol
All steroid hormones are derivatives of what type of lipid?
Cholesterol
What molecule makes up cholesterol’s head group?
OH (it’s very small)
Why do membranes need cholesterol?
Because it decreases local membrane fluidity by tightly binding adjacent hydrocarbons close to the polar head
Explain how cholesterol is like antifreeze
At high concentrations, cholesterol’s bulkiness prevents “freezing” between fatty acid chains
Cholesterol’s strange physics makes it like antifreeze. It prevents the membrane from freezing when temperatures are low, but it prevents it from boiling and dissociating when temperatures are high
Cis-double bonds (fatty acids)
Creates inflexible kinks
Trans-double bonds (fatty acids)
Straightened double bond, no kinking.
Cannot be metabolized by anything because they aren’t found in nature, thus giving them a super long shelf life
Hydrogenated fats
Polyunsaturated fatty acids that have had a H forced onto its kinked double bond to saturate it and straighten it out. Hopefully this process creates a cis-double bond, but sometimes trans-double bonds can be created instead (trans fats)
Membranes with more saturated tails
Floppier, can be packed together tighter, makes the membrane less fluid
Membranes with more unsaturated tails
Less floppy, packed more loosely, membrane is more fluid
When discussing a fatty acid, always consider…
- Its length, aka how many carbons there are in its chain
- Its saturation vs unsaturation level
- The placement of its unsaturations, omega-3 vs omega-6
The cytosolic leaflet has different lipid ratios than the extracellular leaflet. What are the implications of this?
Exterior lipids play roles in normal cell signaling
Phosphatidylserine stays on the exterior side, but during apoptosis it flips to the cytosolic side. This signals to other cells that this guy is undergoing apoptosis, and that they need to gobble this guy up. It’s an “eat me!” signal
ATP-dependent flippases are needed to move a lipid from one leaflet to another. They use ATP hydrolysis to do so. Their function is to cover the hydrophilic head group so the lipid can be passed through the hydrophobic interior of the membrane to the other leaflet
A typical eukaryotic plasma membrane is ~50% proteins by weight, but the actual protein:lipid ratio is 1:50. Why?
Because the proteins are so much bigger than the lipids
How many protein coding genes do humans have?
20,000
How many of our (human) protein coding genes code for membrane proteins?
7,000
List three examples of membrane protein functions
- Proteins that regulate passage and transport through the membranes
- Cell surface receptors
- Localization, i.e. protein surfaces used to hang things up on like paintings on a wall
List 3 major ways a protein can associate with a membrane
- Integral (part of the protein is in the lipid bilayer)
- Ligand linked (protein is post-transl. modified to covalently link to the membrane’s lipids)
- Peripheral (proteins are non-covalently bound to an integral membrane protein)
Integral membrane proteins
Part of the protein is in the lipid bilayer
Must have at least one TMD
What tertiary structure are most TMDs made of?
Alpha helices, which must be 20-25 aas long to span the membrane (3.5 nm)
Beta barrels are much rarer
Glycophorin A
Example of an integral membrane protein
Contains a single alpha-helix TMD
Forms dimers in RBC membranes
This is the protein to which the parasite that causes malaria binds to
Beta-barrels as integral membrane proteins
Beta barrels are rare to TMDs, if present they must be 10 aas long to span the membrane
Can function as receptor ligases for ion transport
Large barrels will function as pores in the membrane
Ligand linked proteins
Proteins have been post-transl.modified to covalently link to the membrane’s lipids
How are ligand linked proteins linked to the membrane?
Cytosolic enzymes catalyze the formation of the covalent linkage
What happens when the formation of the linkage between a ligand linked protein and its membrane is blocked? Aka what happens when the prenylation does not occur?
RAS example
RAS is a lipid linked signal transduction protein common to cell proliferation and cancer when dysregulated.
Researchers KO’d its prenylation enzyme ICMT in mice. The results were that RAS no longer had its transmembrane domain, and was subsequently found all over the cytosol instead of only in the membrane.
This broke the mice’s signal transduction pathway and increased their risk of developing cancer
Peripheral membrane proteins
Associated to the membrane by non-covalently binding to an integral membrane protein within the membrane
How do lipids within a membrane interact with a surrounding membrane protein?
The lipids can be restructured and/or immobilized by interaction with the protein
Similar to how water orients itself around dissolved ions
Hydrophobicity plots
Used to predict whether a protein is an integral membrane protein using its primary sequence.
It takes amino acid chunks of a protein (ABCDE, BCDEF, CDEFG, DEFGH, etc) and calculates the energy needed to move each chunk from a nonpolar solvent to an aqueous medium.
Nonpolar chunks will need hella energy, while polar chunks will release energy.
This is plotted in a graph, the so-called hydrophobicity plot. X = amino acid residue, Y = deltaG.
What type of TMDs are hydrophobicity good at picking up?
Alpha-helix TMDs
What type of TMDs are hydrophobicity plots bad at detecting?
Beta barrels
Because the amino acids in a beta barrel alternate hydrophobicity every other, it won’t show up on this particular test.
If you lyse open a cell and pellet the plasma membrane, what proteins could be removed with only increased salt?
Peripheral proteins. Salt interferes with ionic bonds and Hydrogen bonds, and it outcompetes the peripheral proteins attachments to the membrane
If you lyse open a cell and pellet the plasma membrane, which proteins could be removed with only detergent?
Transmembrane proteins and other lipid-linked proteins
If you lyse open a cell and pellet the plasma membrane, what might be impacted by a mutation that altered a glycine or a cysteine?
Only certain amino acids can receive the lipid modifications (prenylation?). Glycine and cysteine are two of those amino acids, so it would mess with their lipid modifications (prenylation?)
Why are ionic detergents great for SDS PAGE gels but not for all situations?
Because they completely unfold proteins, especially when heated
Which detergent should you use to remove proteins from a membrane? (Ionic or non-ionic)
Non-ionic
They’re still polar, but not charged. They bind to hydrophobic residues and outcompete the bound surrounding lipids while solubilizing the protein. You do need hella detergent molecules to achieve this tho
How are membrane proteins studied? (general technique) (used a lot by neuroscience girlies)
Membrane proteins are isolated then inserted into a simpler system for analysis.
Cell is lysed, membrane is pelleted, membrane proteins are removed using a non-ionic detergents.
The membrane proteins are then incorporated into simple phospholipid vesicles of known lipid composition.
What is FRAP used for?
Used to quantify the rate of mobility of specific fluorescently labelled proteins
What does FRAP stand for
Fluorescence Recovery After Photobleaching
Are membrane proteins mobile within the cell membrane?
Yes
(Question was first posed by Frye and Edidin in 1970, they used FRAP to prove that yes they do move)
Based on the equation, do large proteins have more or less mobility in a membrane?
Less
Based on the equation, do small proteins have more or less mobility in a membrane?
More
Tight junctions in intestinal epithelial cells
The tight junctions between adjacent cells are needed to prevent the stomach contents from leaking. This gives the cells a polarized character, where some proteins are only found on the apical side, and others are only found on the basal and lateral sides.
List 4 factors that restrict the movement of proteins in a membrane
- Proteins are linked into a complex, preventing them from moving as quickly as they would if they were single proteins
- A ligand, surface, or some other extracellular matrix anchors the membrane proteins in place
- Cytoskeletal structures or other intracellular features anchors the membrane proteins in place
- Cell-cell connections, like the tight junctions in intestinal epithelial cells, prevents the diffusion of proteins throughout the plasma membrane
If you were to FRAP a membrane protein bound to an extracellular ligand, would the area recover?
NO
If you were to FRAP a membrane protein linked to a cytoskeletal structure, would the area recover?
NO
If you were to FRAP a membrane protein involved in a cell-cell connection (like a tight junction), would the area recover?
NO
If you were to FRAP a membrane protein linked in a large protein complex, would the area recover?
NO
If you were to FRAP a membrane protein not bound to any other complex or proteins (one that’s just free in the membrane), would the area recover?
Yes
What substances are the best at diffusing across lipid membranes?
Gasses (think diatomic nonpolar ones, like O2, N2, H2, etc) and lipids
These two (for the most part) don’t use channels or transporters. They just do simple diffusion through the plasma membrane to enter the cell
What substances are the worst at diffusing across lipid membranes?
Ions (highly charged = highly hydrophilic)
How good are uncharged, nonpolar, and hydrophobic molecules at diffusing across lipid membranes?
Very good
Second best, only outranked by gasses and lipids
How good are small, uncharged, polar molecules at diffusing across lipid membranes?
They’re okay
How good are large, uncharged, polar molecules at diffusing across lipid membranes?
They’re not good
How good are ions at diffusing across lipid membranes?
Dogshit
Why do lysosomes have a ~100x higher H+ concentration inside them?
The high pH in lysosomes supports its acid hydrolases, the enzymes that break down stuff (the purpose of the lysosome) These enzymes can only function at highly acidic pHs
Why is establishing and maintaining large ion concentration gradients important? (general answer)
It’s key to cell function
K+
Where is it more concentrated, intracellular or extracellular?
Intracellular
Cl-
Where is it more concentrated, intracellular or extracellular?
Extracellular
Na+
Where is it more concentrated, intracellular or extracellular?
Extracellular
Ca2+
Where is it more concentrated, intracellular or extracellular?
Extracellular
K+
Where is it less concentrated, intracellular or extracellular?
Extracellular
Cl-
Where is it less concentrated, intracellular or extracellular?
Intracellular
Na+
Where is it less concentrated, intracellular or extracellular?
Intracellular
Ca-
Where is it less concentrated, intracellular or extracellular?
Intracellular
Cellular spending money
NADH, FADH2, ATP
Harvested from the chemical bonds in food/fuel like glucose, fats, and amino acids
Cellular battery power
Potential energy
Created with large ion concentration gradients across membranes
How much more PE does a cell membrane have than a high voltage power line?
10^5 times more PE
Cell membrane has 200k volts/cm
High voltage power line has 200k volts/km
5 fold difference
Are transporters active or passive?
Trick question. They can be both, it depends on the transporter
Active transporters
Energy is needed to move molecules UP a gradient
Passive transporters
Molecules naturally move DOWN a gradient, no energy required
Uniporters
Passive transport
Transport involves conformational changes in the transporter
The required shape shifting limits the uniporter’s speed. This gives each uniporter a finite capacity, which can be maxed out
Glucose Transporter 1 (GLUT1)
Passive transport
Used by most cells to take up glucose from the bloodstream
GLUT1 moves molecules DOWN a gradient, from high → low
Does NOT require energy
Transport is ligand-specific
GLUT1 transport rate is higher than unfacilitated passive diffusion
Transported molecules move through a protected space, never actually interacting with the membrane bilayer
Transport involves a series of shape changes, see the image of uniporters above for an example
Transport can run either forwards (glucose in) or backwards (glucose out), depending on the direction of the concentration gradient
What tertiary structures are found in GLUT1 uniporters?
12 alpha-helices
Hydrophobic residues of the proteins are found closer to the membrane’s fatty acid tails
Hydrophilic residues are closer to the cytosol and interior compartment
Cells want to take in more sugar, but passive transport is limited by concentration gradients. How do cells get around this?
Hexokinase immediately converts intracellular glucose to G6P upon entry into the cell. This maintains transport efficiency, and is the first step in glycolysis
To avoid a G6P equilibrium trap, GLUT1 is ligand-specific, and doesn’t bind to G6P. As far as the transporter knows, there’s still a huge gradient of exterior glucose
How many different glucose transporters do humans have?
14
Each has their own distinct kinetics
Is GLUT1 (and other uniporters like it) saturable?
Yes
Vmax, the maximum transport rate, is limited by the number of transport proteins (i.e. the number of GLUT1 transporters)
Km
A mathematical value used to compare glucose transporters to each other.
More efficient transporters have lower Kms, less efficient transporters have higher Kms
What should the Km of a very efficient transporter be?
Low
What should the Km of an inefficient transporter be?
High
What’s the advantage to a transporter having a high Km? Aka, what’s the advantage to being an inefficient transporter?
Different Km values indicate different affinities for glucose, which allows the transporters to be regulated differently
High Km transporters are much more sensitive to a wider solute gradient rage
Low Km transporters are much more saturable than high Km transporters, again allowing for differential regulation
How do we know there’s an advantage to having inefficient (high Km) transporters? What techniques were used to learn this?
- Analysis of isolated glucose transporters in pure phospholipid membranes (lab-made micelle-like structures of known lipid composition)
- Introducing mutated transporters into live cells
- Structure discernment and resolution (based on known primary sequence, of course)
GLUT1 Km value for D-Mannose
20
GLUT1 Km value for D-Glucose
1.5
GLUT1 Km value for D-Galactose
30
Is active transport primary or secondary?
Trick question. It can be primary or secondary, it depends on the transporter.
Note that both types are driven by ion gradient battery power
Primary active transport
ATP-powered pumps
