Pass through membranes; can form channels or serve as receptors for bound molecules on one side, passing the information on to the other side
Peripheral membrane proteins:
Proteins associated with other proteins Associated with the plasma membrane
Lipid anchored proteins:
They're covelently attached to either a phospholipid or a sterol
Used to stain cell, show lipid bilayer
The viewing of proteins or other molecules by way of crystallization and the use of x-rays
Hollow vesicle of phospholipids used to administer drugs in the lab (Can engineer them to have antibodies on the outside for cell targeting)
Protein strongly upregulated on breast-cancer cells
Anti-bodies are often:
Bivalent, but they can be made monovalent
Detecting a phospholipid which should be on the inside leaflet will do what?
Cause cells of the immune system to kill that cell – apoptosis
Proteins responsible for flipping back a misflipped phospholipid in the plasma membrane
Fluorescent recovery after photobleaching; Detects lateral movement of proteins and lipids On the membrane by fluorescent labeling and laser bleaching. Can also detect if a protein is attached to this cytoskeleton and does not move
A Fused hybrid cell to measure the diffusion of proteins On a membrane surface; fluorescently labeled anti-bodies attached to the proteins and the diffusion can be observed
Fluorescence activated cell sorting; Detects the coloration of fluorescently tagged antibodies on the surface of Different cells for the purpose of Separation; Disadvantage – due to use of anti-bodies, certain proteins may not be available to do something else
Impact the permeability and the freezing temperature of membranes; Inserted in the bilayer, preventing the movement of small molecules from passing in between the neighboring phospholipids and stiffening the bilayer, and could freeze at a higher temperature
What increases fluidity and reduces the freezing temperature of membranes?
Unsaturated hydrocarbon tails with cis- double bonds, Reducing stiffness
Temporary assembly of lipid molecules in the membrane Which proteins can then assemble onto and further stabilize the raft and organize membrane proteins; Lipid rafts help to concentrate proteins into microdomains To set up signal transduction cascades so that you can have a signaling molecule interacting with multiple proteins, which and then interact with other proteins in the cell to cause a signal transduction cascade
Atomic force microscopy – a way to see Lipid rafts
Many ways membrane proteins associate with the lipid Bilayer:
Singlepath Alpha helices– a polypeptide passing once Or multiple times
Baby sheets maybe rolled up forming a beta barrel, good for forming pores
Domains of transmembrane proteins:
Extracellular: Nitrogen end
Membrane spanning helices: Hydrophobic and uncharged
Cytosolic domains: carbon end – Positively charged amino acids (arginine, lysine) attracted to negatively charged phospholipid head groups, Holding the protein in place
Identified by hydrophobicity plots
The number of times the protein passes through the plasma membrane indicates:
Its function; passing once, one strand on each side, And only certain molecules can interact with that strand. Multiple passes can allow for a pore. Can be suggested from genome sequence
How to experimentally convert a single chain multi pass protein into a multi chain multi pass protein?
Proteolytic cleavage from a protease
Proteases Cleave in a sequence specific way...?
True… Therefore you will know where it's going to cleave. Then you can study the function of areas the protein
Tag on the end of the proteins that directs it where to go
Channel proteins that have some specificity for the small molecules they transport. Most are trimers of three identical protein subunits, each containing 16 beta strands forming a beta sheet, Resulting in a hydrophilic interior and hydrophobic exterior
How to make a bigger porin?
Add more beta sheets
Not all beta barrels directly diffusion of molecules…?
True, some are so tight they act as a receptor or as an enzyme, Hydrolyzing phospholipids (Phospholipases)
Addition of sugar residues to proteins inside The ER or the GA. Result is attachment of oligosaccharides to the noncytosilic part of the protein, Glycoproteins.
(Many are transported through the Secretory pathway through the ER/Ga, And released out the plasma membrane)
The cytosolic side of the plasma membrane has a reducing environment such that:
cystine residues will not form disulfide bonds. Get these bonds form on
the non-cytosolic side giving proteins their shape for their function.
It also allows for two proteins to come together
One way proteins can be glycosylated differently?
Proteins can be glycosylated differently depending on when it was made. And how their glycosylated will impact their function
How do we study membrane proteins?
Solubilize them using non-ionic detergent, amphipathic and more soluble in water then lipids. Then we can isolate proteins using antibodies, Crystallography, or whatever, put them back into a lipid bilayer, And test them.
What keeps membrane proteins localized on particular regions of the membrane?
Tight junctions, self-assembly of proteins into aggregates, tethering with macro molecules either on outside or inside of the cell.
First multipass (7 Alphahelices) membrane protein. whose function was discovered by identifying it's crystal structure. On the surface of bacteria in Purple Aggregate patches, Color due to interior chromophore. With the light, confirmation of protein's retinal changes, inducing change in protein shape. Allows for proton transfer to Inside of cell For ATP formation
Permeability of molecules across a synthetic membrane:
Small hydrophobic molecules (02, CO2, steroids) dissolve in lipid membranes and fuse across. Small and large, uncharged polar molecules diffuse at a lower rate. Impermeable to large molecules and ions
A.k.a. carriers or permeases, Hey mind to a specific solute undergo a confirmational change and transfer the solute across the membrane
Allow specific solutes to pass through when open; more rapid than using transporters
Occurs spontaneously down an electrochemical gradient, either through simple or facilitated diffusion
Transporters require energy to transport molecules against their electrochemical gradient
The electrochemical gradient is a combination of:
Membrane potential and solute concentration gradient
ATP powered pumps:
ATPase; utilizes energy by ATP hydrolysis, relatively slow
Relatively fast, cannot either being non-gated and open most of the time, or gated, open only in response to specific signals; down gradient
Intermediate speed; you know porters, seem porters, anti-porters, code transporters; can either go down gradient or use ATP hydrolysis
Kinetics of simple and transporter diffusion:
Simple diffusion, linear relationship between velocity and concentration; Transporter mediated diffusion, initial exponential growth reaching carrying capacity
In what three ways do cells use active transport?
Coupling with another solute going down its gradient, using ATP hydrolysis, using light energy (bacteria and Archaea)
Transporting down gradient of a single solute alternating between two confirmational states. Ex: Glut one imports glucose
Glucose entry by symporter:
Goes against gradient by coupling with Na going down gradient. Oscillates between a and B states
Trans-cellular transport of glucose:
Glucose pumped by na-powered glucose symporter through apical plasma membrane, passed through Uniporter at basil domain, where Na-K anti-porters keep cytosolic Na low
Four types of ATP driven transport pumps:
P-class pumps, v-class proton pumps, f class proton pumps, ABC superfamily
P class pumps:
Phosphorylate themselves, transport ions(Sarcoplasmic reticulum calcium pump)
Cavity for two calcium ions on cytosolic side, ATP binding and phosphorylase and causes rearrangement, disrupts and releases calcium on the other side, Causes dephosphorylation and another conformational change.
Anti-porter, sodium potassium pump:
Functions similarly, but pumps into potassium for every three sodium pumped out of cell – energy Intense (When experimentally reversed, can synthesize ATP)
V and F class proton pumps:
Functions similarly to P class pumps
Flipase and MDR1 (Multi drug resistance protein, actively transport drugs out of cancer cells major problem)
Pump small molecules with two ATP binding/catalytic domains or cassette
Hydrophilic amino acids lining one face of the pore cause water molecules to pass in single file due to transient hydrogen bonds, helped by hydrophobic amino acids on other side. Two Asparagines in Center attract two molecules in unison inducing them to turn
Ion channel proteins:
Are gated, with ion selectivity, passive. Polar amino acids line the channel, hydrophobic ones interact with the membrane
Four types of stimulus regulating ion channel gates:
Change in voltage across a membrane; binding of the ligand to that gated ion channel; mechanical stress; phosphorylation/Dephosphorylation
Selectivity filter of the bacterial potassium Channel:
Water is stripped off the ions as they pass through the selectivity filter which requires energy. However, lost energy is balanced by lining carbonyl oxygen. Similar to aquaporin concept
Potassium channel regulation:
Can be the voltage depending confirmational change that moves the Alpha helices in the membrane. Depolarization opens, hyperpolarization obstructs
G coupled receptors:
Receptor receives Neuro transmitter gives G protein inside the cell to a neighboring effector protein which then signals ligand on an ion gated channel
Where do intracellular signals regulating ligand gated ion channels come from?
Membrane bound organelles and extracellular environment
Instrument used to measure electric potential across the membrane; patch clamp: can measure a single ion channel and time it precisely
Action potential across neuron membrane:
Rapid activation and inactivation of sodium channel along with activation of potassium channel, thereafter slow inactivation
Electric'stimulus Depolarizes part of membrane (equal charge on both leaflets), causes sodium channels to open, sodium flows in, Increases depolarization, neighboring sodium channels open so on. Eventually potassium channels open, allowing potassium to flow out of cell and stop localized action potential.
Rapid inactivation of potassium channels – ball and chain model:
Depolarization causes channel to open, but remaining depolarized adopts the inactive confirmation plugs the pore, helps to repolarize membrane
Budding and fusion– maintains orientation of lipids and proteins
Guide for protein transport:
Nucleus has gated transport with cytosol; transmembrane transport from cytosol to peroxisome's, plastids, mitochondria, ER; Vesicular transport between ER and GA, and GA to endosomes, lysosomes, secretory vesicles and cell exterior
Nuclear pore complex:
Permeable to very small molecules, most often use transport. Yet pores in NPC are much larger than transloconpores, because proteins go through fully folded
F-G nuclear porins:
Gate-forming proteins of the NPC
Nuclear localization signals:
Direct active gated transport of proteins into the nucleus, Without it they remain in cytosol; cytoplasmic proteins Fuse to NLS, Visualized using immuno gold electrons microscopy (mutated NLS prevents nuclear transport)
Mechanism of nuclear import:
NLS on a cargo protein binds to a nuclear import receptor, Inducing change in the receptor, causing interaction with the F – G nuclear Porins. Entry into nucleus. RAN in the nucleus, when bound to GTP, has a higher affinity for the nuclear import receptor than NLS. RAN GTP attaches Two new site, releasing cargo proteins from old; receptor conforms and exits. In the cytosol, RAN GAP hydrolyzes RAN GTP to GDP, Releasing RAN from the receptor. Then renters and RAN – GEF (Attached to chromatin) exchanges that RAN GDP for GTP again.
Mitochondrial protein transport:
Proteins moved into mitochondria in linear form after being fully translated from the ribosome. Most destined for that Matrix have an N-terminal signal peptide and must cross two membranes, Usually simultaneously.
Chaperones unfolds protein, exposing a matrix targeting sequence, Which binds to import receptor on outer membrane, TOM 22. Signal peptide is transferred to Tom 40, Which has pore to move proteins through the membrane. If it has signal to target it to inner matrix, It then goes through TIM 23 as well. Protease Cleaves signal sequence, and matrix chaperonins refold the proteins
Movement of proteins into the ER occurs Cotranslationally...?
Three ways for protein transport into inner mitochondrial membrane:
After signal sequence cleaving in matrix, it exposes a hydrophobic stop – transfer sequence. Hydrophobic amino acids prevent proteins from being further transported through Tim
Oxal targeting sequence:
Tim should push through protein, signal sequence is Cleaved, Second signal sequence bring protein to another translocon, SS Binds onto oxal1, forwards to Intermembrane.
Chaperones – directed transfer to TIM 22: Protein binds onto Tom gets pulled in and is released in inner Membrane space Without targeting sequence to Tim Chaperones coat proteins to expose new signal sequence and does join Tim. Requires just one translocon
Ways to get protein into into your membrane space:
One way you requires no proteases and only a single for Tom, the other requires two proteases and a signal for both Tom and TIM
Proteins entry to ER:
Co translational and post translational
Post translational protein entry to ER:
Sec complex recruits binding protein (similar to chaperone in mitochondria) in the ER lumen. Drives ATP dependent unidirectional translocation of the peptide into Lumen. In other words BIP binding and release of proteins pulls it into the ER.
Cotranslational translocation into the ER:
N-terminal ER signal sequence finds it to S RP (signal recognition particle). Direct the ribosome to SRP receptor. Ribosome transferred to trans low con. It opens, SRP dissociates the GTP hydrolysis; proteins passes through trans locater and into blooming. Signal sequence cleaved. Once translation is complete ribosome dissociates
Glycosylation in rough ER:
Upon entering, most proteins are immediately N– glycosylated on target asparagine amino acids. Mediated by ER membrane bound oligosaccharal transferase enzyme
Sugars add to keep Protein in ER until it is completely folded. If it can't be folded it will be exported and degraded by Proteosome
Simple intracellular signaling pathway:
Signaling molecule binds receptor on surface of self, induces confirmational change in the receptor, the change in receptor shape is propagated in amplified inside the cell by signaling proteins, results in changes in metabolism in gene expression Or cell shape. Mediated by effector proteins
Five types of intercellular signaling processes:
Contact dependent, paracrine, synaptic, endocrine (distal), gap junction
Endocrine versus neuron signaling:
Both transport long distance signals, but endocrines releases lipophilic hormones which are stable and transported through circulation systems. Neurons release hydrophilic molecules (neurotransmitters) that are transiently stable and transported To neighbors
Alterations in protein function or Gene transcription:
Intracellular signaling pathways to produce altered protein function WorkFast. So surface receptor proteins to alter proteins synthesis in the nucleus are slow
The same signaling molecules binding different cells can have different effects on cellular processes…?
Intracellular receptor, nuclear receptor and steroids ligand:
Three domain proteins when bound to ligand induces confirmational change in receptor – transcription either stimulated or repressed
Generalized intracellular signaling pathway:
Signal first transduced, relayed, amplified, integrated. Proteins organize components of signaling pathway. Anchors localize components, other Components can regulate intensity of signal
Two molecular switches regulating signals in the cell:
Add addition of phosphate by Kinase to signaling proteins; exchange Of GDP for GTP
Positive versus negative feedback for addition of phosphate by Kendes and removal by phosphatase:
With positive feedback, kinase activity barely lowers; with negative feedback it drops and drops again
G – protein coupled receptor. Largest family of receptors, binding ligands and transfer signals to the interior of the cell. Relays signals to trimeric G proteins, a single chain Crossing membrane seven times.
Molecule activate GPC are
GTP binding and hydrolyzing trimer protein, attached to inner plasma membrane, Couples G PCR to enzymes or Ion channels. Alpha helices is the GEF factor, beta sheet, G TPAse
Activation of g proteins by GPCRs:
Extracellular signal causes confirmational change in G PCR, induces change in G protein, changes alpha subunits the G protein to exchange GDP for GTP, and receptor stays active so long as ligand is bound to G PCR
Intracellular messenger made by Adenylyl cyclase in response to ligand binding a GPCR
Camp binding to camp – dependent protein kinase A (PKA):
Releases catalytic subunits that affect transcription
G PCR – camp process:
Signal molecule activates G PCR activates Adenylyl cyclase attached to alpha subunit of the G protein. Activation and confirmation of G protein causes production of camp With use of ATP. Camp binds to regulatory subunits to activate PKA, PKA enters nucleus, activates transcription factors (inactive CREB), result is changing phenotype
How is camp rapidly degraded?
In cell by camp phosphodiesterase
Many G proteins activate plasma membrane bound phospholipase C – Beta:
PLCB typically associated with the inner plasma membrane, converting phospholipids into fatty acids
Another secondary messenger generated by phospholipase activation from activated G protein. Rapidly diffuses through cytosol, opens IP three gated calcium channels. Rapid rise in cytosolic calcium, activates calcium sensitive proteins like protein kinase C and calmodulin
Specialized ER, stores and releases calcium in response to electric signals. Causes membrane to depolarize resulting in muscle contraction
Most important calcium binding proteins, highly conserved, relays cytosolic see a signal, regulator of CA processes, has four-calcium binding domains. Two or more findings activate calmodulin – no catalytic activity it's self, it binds to and activates other proteins, like kinase
CAM – kinase activation:
Three domains: kinase, regulatory, and calmodulin interaction. Cam kinase II: Molecular memory device, activity increases with high-frequency stimulation by oscillation of calcium/calmodulin and protein phosphatase. Hyper activated enzyme can remain highly active in absence of calcium, important for memory in Neurons
Signal molecule activates cytosolic RT case for phosphorylation And activates signaling proteins relay downstream. But with mutant tyrosine kinase Domain, no kinase activation occurs
More direct way for activated cell receptor to impact transcription. Uses transforming growth factor b. Dimerization causes phosphorylation and then recruits and phosphorylates smad2/3, which binds smad 4, translocate's complex to nucleus, and binds TGF beta response affecting gene transcription