BCHEM UNIT 2 - 3 (lipids)

Unlike phospholipids which move at a uniform rate, how do proteins differ in their rates of lateral diffusion?

-proteins differ in their rate of lateral diffusion; some move quickly and some are immobile -rate of protein mobility depends on its function and whether or not its anchored

Describe the asymmetry of membrane lipid bilayer composition What is a reason that helps explain this asymmetry

-the 2 sides of the membrane bilayer are structurally/functionally different 1) Signal Transduction = different lipids act as different binding sites for proteins/enzymes/signals

How are steroids/retinoids/thyroids unique as hormones? Describe the 4 step mechanism:

-they act in the nucleus to alter gene expression MECHANISM: 1) hormone carried to target tissue on serum binding protein; it diffuses across plasma membrane and binds to receptor in nucleus 2) binding changes conformation of receptor to either homo/hetero-dimeric complex and then binds to HRE (hormone response element) in the DNA next to a specific gene 3) complex attracts co-activator/co-repressor and regulates transcription of adjacent gene, increasing or decreasing rate of mRNA formation 4) cellular response based on gene product

Describe the model of glucose transport: -2 conformations -4 steps

-transport exists in 2 conformations: 1) T1 = binding site on outside 2) T2 = binding site on inside STEPS: 1) glucose in blood binds to T1 2) binding lowers Ea for conformational change from T1 to T2 3) glucose released from T2 into cytoplasm 4) transporter returns to T1

What are the 3 general classes of transport systems?

1) Antiport = 2 substrates moving in opposite directions 2) Symport = 2 substrates moving in same direction 3) Uniport = 1 substrate being moved

What are the 7 major functions of biological membranes?

1) Define external boundaries 2) Control molecular traffic 3) Divide cells/organelles 4) Organize rxn sequences 5) Cell-cell communication 6) Transporters/adhesion 7) Energy transduction within cell

What are the 4 general types of receptors found in signal transduction? Which 2 take place in the nuclear envelope (as opposed to the plasma membrane)?***

1) G-PROTEIN COUPLED = external ligand (L) binds to receptor (R) and activates an intracellular GTP-binding protein (G) which regulates an enzyme (Enz) that generates an intracellular second messenger (X) 2) ***TYROSINE KINASE = ligand binding activates tyrosine kinase activity via autophosphorylation, which activates transcription factor (T) to alter gene expression 3) GATED ION CHANNEL = channel opens/closes in response to [L] OR membrane potential (Vm) 4) ***NUCLEAR RECEPTOR = hormone binding allows the receptor to regulate the expression of specific genes

Describe the 7 step mechanism by which RTKs operate

1) INSR binds insulin and undergoes autophosphorylation 2) INSR phosphorylates IRS-1 (target protein) on its Tyr residues 3) Grb2 binds to P-Tyr of IRS-1; SOS binds to Grb2, then to RAS, causing GDP release and GTP binding to RAS 4) Activated RAS binds/activates RAF-1 5) Protein Cascade; RAF-1 phosphorylates 2 Ser residues on MEK, which in turn phosphorylates ERK on Thr & Tyr 6) ERK moves into nucleus and phosphorylates ELK-1 (nuclear transcription factor) 7) ELK-1 joins SRF to stimulate transcription/translation of a set of genes needed for cell division

Define the 3 types of membrane proteins and: -type of binding -method of removal

1) INTEGRAL = firmly inside bilayer -Binding = hydrophobic interactions -Removal = anything that messes with hydrophobic interactions (detergent, denaturants) 2) PERIPHERAL = rests on top (outside) of bilayer -Binding = electrostatics/H-bonding -Removal = anything that messes with the above a) changing pH b) mild detergents c) adding carbonate 3) AMPHITROPIC = reversible binding; found in both cytosol/membranes -Binding = covalent/non-covalent -Removal = regulated reversible binding

What is the difference in lateral diffusion vs. flip flopping of lipids and the speeds at which they take place? What explains the speed at which they take place? Which one typically requires catalysis?* What 2 types of enzymes are required?

1) LATERAL DIFFUSION = movement of phospholipid/protein in plane of the bilayer which helps maintain membrane FLUIDITY -rapid because no energy barriers are required to overcome 2) *TRANSVERSE DIFFUSION (flip flop) = phospholipid switches leaflets -slow & energetically unfavorable because: a) stabilizing bonds between H2O and polar head are broken b) hydrophilic head travels through hydrophobic core of bilayer

What are the 2 types of Ion Channels:

1) Ligand-Gated = binding of small molecule forces an allosteric transition in the protein 2) Voltage-Gated = change in transmembrane potential (Vm) causes a charged protein domain to move relative to the membrane, opening or closing the channel

What are the 3 types of reversible binding regarding Amphitropic membrane proteins?

1) Lipidation = covalent 2) Phosphorylation 3) Ligand Binding

What is the function of epinephrine? What are its 2 synthetic analogs and their structures/functions? Out of all 3, which has the lowest* affinity for the receptor? Which has the highest***?

*EPINEPHRINE = biological ligand produced by adrenal glands ISOPROTERENOL = Agonist (binds to receptor and mimics effect of natural ligand) ***PROPANOLOL = Antagonist (bind without triggering normal effect & block agonists) -Low Kd = high affinity (steep curve)

What is a Hydropathy Plot? Define the following for the Hydropathy Index: -definition -ΔG > 0 -ΔG < 0 Compare the Hydropathy Plots for Glycophorin vs. Bacteriorhodopsin. What does each peak signify?

-Analyzing AA sequences to predict membrane protein secondary structure -Hydropathy index (hydrophobicity of residue) is plotted vs. residue number. -ΔG > 0 = HYDROPHOBIC -ΔG < 0 = HYDROPHILIC -each peak signifies a segment; the # of UPWARD peaks signifies the number of transmembrane (hydrophobic) segments

What are the 2 types of transporter proteins? Define for them the following: -structure -stereospecificity -rate of transport -saturable?

CARRIER PROTEIN: -alternates between conformations -Stereospecific = highly -RoT = low -saturable CHANNEL PROTEIN -water filled pore across the bilayer -Stereospecific = low -RoT = high -not saturable

In regards to FREE FATTY ACIDS, how do the following affect MELTING POINT? -chain length -degree of unsaturation (cis vs trans) Explain why.

CHAIN LENGTH: -↑ chain length = ↑ van der waals = ↑ MP DEGREE OF UNSATURATION -↑ cis bonds = ↓ van der waals = ↓ MP -trans fatty acids have higher MPs than cis, but still less than saturated

Define the following for Bacteriorhodopsin: -classification -structure -function Bacteriorhodopsin is a trimer. What does this mean?

CLASSIFICATION = Type III STRUCTURE = 7 hydrophobic internal α-helices (non-polar AAs) interacting with nonpolar hydrocarbon tails FUNCTION = uses light to establish proton gradient via light absorbing pigment (retinal) buried inside membrane -trimer is a polymer comprised of three monomers

What are the 3 essential components of G-Protein Coupled Receptors?

COMPONENTS: 1) Cell-Surface Receptor = integral receptor on plasma membrane with 7 TM helices 2) Effector enzyme = enzyme in plasma membrane that generates an intracellular 2nd messenger 3) G-Protein = activates the effector enzyme

What is autophosphorylation? Describe the 3 step method of autophosphorylation in the 1st step of RTK operation

AUTOPHOSPHORYLATION = phosphate added to a protein kinase by itself 1) insulin binds to INSR and activates catalytic domain of β-units (intracellular) 2) each β-unit on the INSR phosphorylates 3 Tyr resides near carboxyl terminus of the other β-unit by transferring phosphoryl group from ATP to -OH of Tyr residues 3) this opens up active site so enzyme can phosphorylate Tyr residues of other targets proteins (IRS-1)

What are 6 specific transmembrane classifications that fall under these general groups?

BITOPIC: -Type I = amino end OUTSIDE bilayer -Type II = amino end INSIDE bilayer POLYTOPIC: -Type III = single polypeptide (amino end outside) -TYPE IV = multiple polypeptides MONOTOPIC: -Type V = held to bilayer by covalently linked lipids BOTH MONOTOPIC & BITOPIC -Type VI

A 2nd class of GCPRs is the Phosphatidylinositol System; define for them the following: -effector enzyme -(3) 2nd messengers -(2) target proteins activated

EFFECTOR ENZYME = phospholipase C (PLC) 2ND MESSENGERS: -Inositol Trisphosphate (IP3) -Diacylglycerol (DAG) -Ca2+ TARGET PROTEINS ACTIVATED -IP3 - gated Ca2+ channel (raises cytosolic [Ca2+]) -Protein Kinase C (brought about by increase [Ca2+] in cytosol)

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Ether-linked Lipids Waxes Cholesterol Cholesterol Derivatives Prostaglandins = enzyme on inside of ER membrane

What are the 3 defining features of ABC transporters? As an example of ABC transporters, what is the function of the MDR1? What happens during overexpression.

FEATURES: 1) 2 nucleotide-binding domains (NBDs) inside 2) 2 TM domains 3) one ATP hydrolyzed per molecule of substrate transported MDR1 (multi drug transporter) = confers drug resistance to anti-tumor drugs by pumping them out of the cell to prevent accumulation in tumor (BAD) -overexpression of MDR1 is associated with treatment failure in cancers

What is the Fluid Mosaic Model?

FLUID MOSAIC MODEL = plasma membrane is a fluid combination of 5 things: Internal = phospholipids, proteins, cholesterol External (carbs) = glycolipids, glycoproteins

How is a lipid raft formed? What are their 3 defining characteristics? Where are they typically found? In what 2 ways do they affect membranes?

FORMATION = stable associations of cholesterol and sphingolipids in outer leaflet produce a microdomain CHARACTERISTICS: -slightly thicker -more Lo state (more ordered, less fluid) -more difficult to dissolve with detergents LOCATION: -rich in GPI-anchored proteins in outer leaflet 1) decreases fluidity 2) increases resistance to phase change

Give the following for GLYCEROPHOSPHOLIPIDS: -function -structure -what are the types based on

FUNCTION = membrane support STRUCTURES: (AMPHIPATHIC) -glycerol -2 FAs (nonpolar) - PO4 + X-group substituent (polar) TYPES = based on acidity of X-group substituent and whether or not it carries a charge -type of charge on X-group determines NET charge which results in different properties

What is the overall function of the AE Protein? Which required anions are antiported? What is the enzyme used? Describe the 4 steps in transport and their respective locations.

FUNCTION = to increase rate of bicarbonate buffer diffusion into blood cells to aid in the release of CO2 -simultaneous movement of bicarbonate ion & chloride ion ENZYME = carbonic anhydrase STEPS: Respiring Tissue: 1) CO2 enters and is converted to bicarbonate ion 2) HCO3- exits to blood (Cl- comes in) Lungs: 3) HCO3- enters (Cl- leaves) 4) HCO3- converted to CO2 and is exhaled

What is the function of PKA (protein kinase A)? What are the 3 primary structures in PKA?

FUNCTION = to mobilize glycogen stores in muscle/liver in anticipation of energy needs STRUCTURE: 1) 2 Catalytic (C) Subunits = contains ATP binding site & substrate binding cleft that holds the: -inhibitor sequence of R (inactive) OR -the protein substrate binding site (active) 2) 2 Regulatory (R) Subunits = each R binds to 2 cAMP; causes conformational change that moves inhibitor domain of R out of substrate binding cleft in C 3) A-Kinase Anchor Protein (AKAP) = multivalent protein adaptor that binds a PKA to a specific structure in a cell

Define for the following type of ABC Transporter: [Na+-Glucose Symporter] -function -starting/ending positions of Na+ -2 driving forces -mechanism of glucose transport in epithelial cells (3 steps)

FUNCTION = to take up glucose from intestine into epithelial cells STARTING = intestinal lumen ENDING = blood DRIVING FORCES: 1) high intestinal [Na+] 2) membrane potential (negative inside epithelial cell which draws Na+ in) MECHANISM: 1) glucose transported with Na+ across symporter into epithelial cell 2) moves through cell and passes through GLUT2 passive uniporter on basal surface, and into blood 3) [Na+K+ ATPase] pumps Na+ outward to maintain the Na+ gradient that drives glucose uptake

Give the following for FREE FATTY ACIDS: -function -structure -types What is a driving force imposed on lipids in an aqueous environment?

FUNCTION = used for fuel STRUCTURE = Polar End (COOH group) + Non-Polar Hydrocarbon Chain TYPES: 1) Saturated (solids) = no double bonds (saturated w/ H's) 2) Unsaturated (liquids) = at least one double bond; can be cis or trans -HYDROPHOBIC EFFECT (due to nonpolar hydrocarbon chain)

Give the following for TRIACYLGLYCEROLS (TAG): -3 functions -structure How can we tell whether it will be solid/liquid?

FUNCTIONS: 1) energy production (stored in adipose cells, carry stored energy without H2O because hydrophobic) 2) heat production (brown fat) 3) thermal insulation (adipose tissue) STRUCTURE = 3 fatty acids (can be different) + glycerol (linked via ester linkage) -depending on types of FAs linked (sat [solid] vs. unsat [liquid])

What is the general equation used for calculating the amount of energy required for active transport? WHAT is the constant? How is this different for simple transport of an UNCHARGED solute? WHAT explains this? How is this different for simple transport of a CHARGED solute? WHAT are the special variables?

GENERAL: ΔG = ΔG' + RT ln([P]/[S]) R = 8.315 UNCHARGED SOLUTE: ΔGt = RT ln(C2/C1) -C1 = initial concentration (where its coming from) -C2 = final concentration (where its going) -standard free energy change is 0 because NO bonds are made or broken CHARGED SOLUTE: ΔGt = RT ln(C2/C1) + ZFΔΨ -Z = charge -F = 96,480 (Faraday constant) -ΔΨ = membrane potential in mV; even though value is negative, use positive

What is special about GLUT4? Describe its 4 steps to glucose intake

GLUT 4 = insulin dependent transporter -its activity increases when insulin signals high blood glucose concentration

What is the Glycocalyx? What are its 3 primary functions?

Glycocalyx = carb layer of glycolipids found on outside of cell membrane; HIGHLY hydrophilic and so interacts with water in cellular environment FUNCTIONS: 1) Cell Recognition/Adhesion = proteins in surface of other cells binding to carbs of glycocalyx 2) Healing = blood clotting, immune response 3) Nerve Impulse Transmission

What are 3 differences between Ion Channels and Ionophores?

In regards to ion channels: 1) they have a much higher rate of transport 2) they are not saturable (rates don't approach Vmax @ high [substrate]) 3) they are gated in response to some cellular event (ligand or voltage)

What is Membrane Fluidity defined as? What are the 3 factors that affect it?

DEFINITION = the relative movement of lipids as a result of the strength of the intermolecular interactions between the lipids (stronger interactions = more rigid membrane) 1) TEMPERATURE: ↑ temp = ↑ fluidity (relative to its melting temp [Tm]) 2) FATTY ACID COMPOSITION: a) ↑ length = ↑ strength = ↓ fluidity b) ↑ unsaturation = ↑ fluidity (as a result of lower Tm) 3) CHOLESTEROL: a) ↑ cho = ↓ fluidity (as a result of lipid rafts) b) presence of cholesterol helps membrane maintain fluidity over broad range of lipid composition (temp)

What are the 2 contributing definitions for a lipid?

DEFINITIONS: -insoluble in H2O -soluble in organic solvent

What are the 6 types of solute transport across intact membranes?

DOWN CHEMICAL GRADIENT 1) Simple Diffusion (nonpolar solutes move through phospholipids) DOWN ELECTROCHEMICAL GRADIENT 2) Facilitated Diffusion (larger solutes move through protein channel) 3) Ion Channel (charged solutes move through gated protein channel) 4) Ionophore (molecule facilitates transport of charged solute) UP ELECTROCHEMICAL GRADIENT 5) Primary Active Transport (solute moved via energy provided by ATP) 6) Secondary Active Transport (endergonic (uphill) transport of one solute coupled to exergonic (downhill) flow of different solute)

Define the 3 following types of lipid aggregates that form in water and give the following: -shapes -individual unit composition (shape and types)

MICELLE = spherical form; units are wedge-shaped (cross section of head bigger than chain) 1) fatty acids 2) lysolipids BILAYER = 2D sheet form; units are cylindrical (cross section of head equals that of chain) 1) glycerophospholipids 2) sphingolipids VESICLE = hollow sphere that;'s the product of bilayer folding in on itself

What are the 3 general groups of integral membrane proteins and their differences?

MONOTOPIC = associated to membrane from one side but doesn't span bilayer completely POLYTOPIC = spans entire membrane; multi-pass BITOPIC = spans entire membrane; single-pass

Define for the following type of P-Type ATPases: [Ca2+ ATPase (SERCA pump)] -full name -type of transport -function -structure (and domains) -mechanism (3 steps)

NAME = sarcoplasmic and endoplasmic reticulum calcium pumps TYPE = primary active FUNCTION = helps maintain a low concentration of Ca2+ in cell cytosol by pumping Ca2+ into the ER lumen STRUCTURE: 1) N-Domain = where nucleotide ATP + Mg 2+ bind 2) P-Domain = contains phosphorylated Asp residue 3) A-Domain = aids in movement of N&P domains to the 2 Ca2+ binding sites 4) M-Domain (T+S) = contains TM helices AND the 2 Ca2+ binding sites MECHANISM: 1) 2 Ca2+ bind to 2 sites on E1 conformation which is exposed to cytosolic side 2) phosphorylation on Asp residue induces conformation change from E1 to E2 to expose Ca2+ binding sites to lumen; Ca 2+ released against gradient due to ATP hydrolysis 3) dephosphorylation returns E2 to E1

Can membrane proteins diffuse laterally and flip-flop?

ONLY laterally, not flip flop

Define the following for Glycophorin: -where is it found? -classification -structure -symmetry

LOCATION = red blood cells CLASSIFICATION = Type I STRUCTURE = 1 α-helices with: -amino group on outside (hydrophilic) -COOH group on inside (hydrophilic) -1 hydrophobic segment in membrane SYMMETRY = asymmetric; amino terminal group is always on outside

What is Signal Transduction? What are the 2 general types of intercellular (outside cell) signal transduction?

SIGNAL TRANSDUCTION = the conversion of information into a chemical process TYPES: 1) Contact-Dependent = signaling molecule not secreted, but is bound to plasma membrane of signalling cell and interacts directly with receptor of target cell 2) Secreted Molecules = 4 different types

SKIP

SKIP

Describe the following for Voltage Gated K+ Channels: -structure -mechanism

STRUCTURE = 4 subunits; each subunit has 4 TM helices (S1-S4) and an additional 2 that help make up the channel (S5-S6) MECHANISM = each S4 helix has 4 Arg residues which act as the voltage sensor and respond to a change in membrane potential (Vm)

Describe the 3-part structure of the G-Protein Describe the 4 step mechanism by which the G-protein activates adenylyl cyclase At which step is a molecule covalently anchored? Describe.***

STRUCTURE = broken into 3 subunits (α,β,γ) MECHANISM: 1) Gs w/GDP = OFF; when in contact with hormone receptor complex (GPCR), GDP is displaced by GTP and turned ON ***2) Gs dissociates into 2 subunits (α & βγ); Gsα activates Adenylyl Cyclase ***-Gsα unit is held to membrane via lipid anchor (palmitoyl group) 3) Gsα turns itself off through intrinsic GTPase activity (converts its bound GTP to GDP and dissociates from adenylyl cyclase) 4) Inactive Gsα reassociates with βγ complex

Describe the following for Ligand-Gated K+ Channels (Aceylcholine Receptors)(AChR): -structure (3 components) -location -mechanism (3 steps)

STRUCTURE: 1) Ligand Binding Domain -has the Acetylcholine Nicotine receptor 2) TM Domain 3) Intracellular Domain LOCATION = neuromuscular junction MECHANISM: 1) AP reaches presynaptic terminal of motor neuron, which activates voltage gated Ca2+ channels 2) Ca2+ triggers acetylcholine release into synaptic cleft and binds to nicotinic AChR 3) causes conformational change, which causes ion channel to open 4) opening of channel causes Na+, Ca2+ & K+ to pass through into myocyte (muscle cell), depolarizing its membrane and causing muscle contraction

Give the following for SPHINGOLIPIDS: -function -structure -2 types

STRUCTURE: (AMPHIPATHIC) -sphingosine (amino alcohol) -FA (amide linked) -polar head group TYPES: 1) Glycosidic Bond = X is a sugar 2) Phosphodiester Bond = X is phosphate

Describe for INS-R the following: -structure -2 key features

STUCTURE = αβ dimer 1) 2 extracellular α-units (insulin binding domain) 2) 2 TM β-units (cytosolic part is catalytic for phosphorylation activity) -each β-unit has 3 critical Tyr residues in cytosol FEATURES: 1) Autophosphorylation 2) Activation of signaling cascades

What type of protein is Adenylyl Cyclase? What is its function? How does it accomplish this?

TYPE = integral protein FUNCTION = to synthesize cAMP (2nd signal) METHOD = utilizing ATP after association with Gsα complex

Define for the following type of P-Type ATPases: [Na+K+ ATPase] -type of transport -function -2 results -difference to SERCA

TYPE = primary active FUNCTION = maintain low Na+ & high K+ in cell RESULTS: 1) 3 Na+ moved out; 2 K+ moved in 2) membrane potential of 50-70mV produced DIFFERENCE = reversible phosphorylation of Asp is coupled to simultaneous movement of both Na+ and K+ AGAINST their electrochemical gradients -responsible for creating membrane potential

What is a membrane transporter protein? When are they used? In what 2 ways do they work? Compare the free energy graph for diffusion with and without a transporter

Transporter = membrane proteins that carries solute across membrane a) polar solute (can't move through hydrophobic core) b) large solute (can't fit through lipids) 2 WAYS THEY WORK: 1) Lower activation energy (ΔG') 2) Remove hydration shell (binding energy counterbalances +ΔG that accompanies loss of water from substrate)

Define the following relating to INSR: a) IRS-1 b) GRB-2 c) SH2 Domain d) SH3 Domain e) SOS f) RAS g) RAF-1, MEK, ERK h) ELK-1, SRF

a) IRS-1 = target protein of INSR b) GRB2 = adaptor protein whose function is to bring together 2 proteins (IRS-1 + SOS) c) SH2 Domain = domain on GRB2 responsible for binding P-Tyr in a protein partner (IRS-1) d) SH3 Domain = domain on GRB2 responsible for binding proline-rich region of SOS e) SOS = protein that binds to both Grb2 & Ras, to enable exchange of GDP for GTP (on Ras) f) RAS = G protein that's active whenits GDP is replaced with GTP; activates protein kinase cascade g) RAF-1, MEK, ERK = the protein kinase cascade; RAF-1 is first to be activated by RAS and activates MEK via phosphorylation, which activates ERK h) ELK-1, SRF = nuclear transcription factors

Define the following relating to PI-3 Kinase: a) PIP-2, PIP-3 b) SH2 Domain c) PKB d) GSK-3 e) Glycogen Synthase f) GLUT4

a) PIP-2, PIP-3 = phospholipids; PIP-2 converted to PIP-3 via PI-3K b) SH2 Domain = location on IRS-1 where PI-3K is bound and activated c) PKB = protein kinase B; responsible for inactivating GSK-3 by phosphorylating it d) GSK-3 = glycogen synthase kinase; inactivates glycogen synthase by phosphorylating it, which slows glycogen synthesis e) Glycogen Synthase = responsible for glycogen synthesis f) GLUT4 = glucose transporter to plasma membrane

What is the primary type of G-Protein Coupled Receptor? Describe the 7 step mechanism

β-ADRENERGIC RECEPTOR 1) Epinephrine (adrenaline) binds to receptor 2) GDP on Gs (G-protein) is replaced with GTP which causes activation 3) Activated Gs (α subunit) moves to adenylyl cyclase (enzyme) and activates it 4) Adenylyl Cyclase catalyzes the formation of cAMP (2nd signal) 5) cAMP activates PKA 6) Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine 7) cAMP is degraded, reversing the activation of PKA

What is a β-Barrel? What is a driving force for formation as opposed to β-Sheets? Give 3 examples of β-Barrels and the # of strands What is a Porin?

β-Barrel = structure for a membrane protein with 20+ transmembrane segments -driven by maximizing hydrophic bonds and intrachain H-bonding (β-sheets don't do this) Porin = β-Barrels that allow polar solutes to cross outer membrane of bacteria (like E. Coli) via PASSIVE DIFFUSION

For TRYACYLGLYCEROLS, how are the ester linkages formed?

3x dehydration

How does AKAP organize reaction sequences? Define the following: -AKAP79 -AKAP250 (gravin)

-AKAP act as scaffolds, holding PKA catalytic subunits in proximity to specific regions within the cell -AKAP79 = binds PKA to Adenylyl Cyclase -AKAP250 (gravin) = holds PKA to plasma membrane

Describe graphically how cholesterol helps membrane fluidity

-maintains rigidity @ high temp -maintains fluidity @ low temp

Some membranes have covalently linked lipids; what is the purpose of these? What are the 4 primary examples and the AAs to which they're attached?

-anchor membrane proteins to the lipid bilayer

What helps to predict the existence of enzymes that catalyze flip flop diffusion? What are these 3 types of enzymes? Function and what family of enzymes they belong to? Which require ATP?**

-asymmetric distribution of lipids 1) **FLIPPASE (P-type ATPase family) = moves aminophospholipids PE/PS from outer to cytosolic leaflet -phosphytidylethanolamine -phosphytidylserine 2) **FLOPPASE (ABC transporter family) = moves phospholipids from cytosolic to outer leaflet -phosphotidylcholine -sphingolipids 3) SCRAMBLASE = moves lipids in either direction toward equilibrium; activated by Ca2+

What is the significance of membrane curvature? What is the 6 step process by which it occurs?

-helps membrane fuse with other membranes 1) Neurotransmitter (v-SNARE) filled vesicle approaches plasma membrane 2) v-SNARE binds with t-SNARE in the plasma membrane via SNAP25 protein and ZIPS UP, which draws the membranes together 3) zipping up causes curvature which causes HEMIFUSION between outer leaflets 4) Hemifusion = inner leaflets of both membranes come into contact 5) complete fusion creates a fusion pore for neurotransmitter release 6) pore widens; vesicle contents are released into cell

Describe the kinetic equation of glucose transport into erythrocytes and 3 important variables

-similar to enzyme reaction RATE EQUATION: (pictured) 1) Vo = initial velocity of accumulation of glucose when [S]in = 0 2) [S]out = substrate (glucose) outside cell 3) Kt = rate constant

Describe the kinetics plot for glucose transport into erythrocytes. What happens when [S]out = Kt? Compare the Kt values for D-glucose vs. L-glucose. What do they mean?

-when [S]out = Kt, the rate of glucose uptake is 1/2 Vmax -D-Glucose has LOWER Kt and therefore GREATER affinity for transport (less is needed to reach 1/2 Vmax)

What are 2 channels meant for rapid transport? Define for them the following: -type of protein -structure -what they transport -location -mechanism

1) AQUAPORINS = passage of water -Type = integral protein -Structure = tetramer comprised of 4 monomers (4 channels) -Location = exocrine glands (sweat, saliva, tears) & kidney (urine production/water retention) -Mechanism = osmotic gradient w/ selectivity filter 2) ION-CHANNELS = passage of K+ ions -Type = integral protein -Structure = tetramer comprised of 4 monomers (1 channel) -Location = in membranes of all cells -Mechanism = gated w/ selectivity filter

Describe the 3 structural components of rod cells

1) OUTER SEGMENT = contains rhodopsin (photoreceptor) and stacks of membranous disks 2) INNER SEGMENT = contains nucleus/other organelles 3) SYNAPTIC TERMINAL = synapses with interconnecting neurons

What are the 2 families of active transporters? What types of proteins are they? How many are there in the body?

1) P-Type ATPases = cation transporters that are reversibly phosphorylated by ATP -integral membrane proteins (8-10 TM helices) -70 in humans 2) ABC Transporter (ATP binding cassettes) = pump solute out of cell against a concentration gradient -integral membrane proteins (12 TM helices)

What are the 4 different methods of Secreted Molecule Signal Transducing Systems? What are the 8 specific types of secreted signalling molecules

1) Paracrine = acts on neighboring cells 2) Autocrine = acts on cells that secret signalling molecule 3) Endocrine = acts on cells that are remote from secreting cell 4) Synaptic = fast signalling involving neurons (nerve cells)

What are 2 other Topology rules?

1) Positive-Inside Rule = + charged AAs usually found inside bilayer (Arg, Lys, His) 2) Tyr & Trp are commonly found at the membrane water-lipid interface for simultaneous interaction

Define the following features of signal transducing systems: 1) specificity 2) amplification 3) modularity 4) desensitization/adaptation 5) integration 6) localized response

1) SPECIFICITY = ability for signal molecule to be specific to receptor's binding site 2) AMPLIFICATION = ability for enzymes activating enzymes, which causes exponential increase of activation 3) MODULARITY = ability for proteins with multivalent (many applications) affinities form diverse signaling complexes 4) DESENSITIZATION = ability of receptor activation triggers feedback that shuts off receptor 5) INTEGRATION = ability for system to receive multiple signals and produce 2 opposite effects depending on metabolic needs 6) LOCALIZED RESPONSE = ability for enzyme that destroys an intracellular message to be grouped with the message producer, causing response to be local and brief

Describe the 3 factors of the selectivity filter for Aquaporins What is the AA involved?***

1) Size Restriction = narrow passageway that restricts large molecules 2) Electrostatic Repulsion = narrow passageway with + charged residues that prevent the movement of ions (protons) 3) Water-Dipole Reorientation = 2 helices in passageway oriented with + dipoles pointed @ pore to force H2O to reorient itself, which breaks up H-bonded chains and prevents proton passage through "proton hopping" -***Asn residues

What are the 4 methods that cause termination of the β-ADRENERGIC RECEPTOR?

1) [L] LOWERED below Kd for receptor 2) Activator proteins (GAPs) stimulate GTPase activity on the Gsα unit (converting GTP to GDP) 3) Hydrolysis of cAMP to 5'-AMP 4) Desensitization Pathway (desphosphorylate activated proteins) via Beta Arrestin (BARR)

What are the 4 steps in the Desensitization Pathway of the β-ADRENERGIC RECEPTOR (the off switch)?

1) after dissociation, βγ complex recruits βARK to membrane, where it phosphorylates Ser residues @ the carboxyl terminus of the receptor 2) βARR (β Arrestin) binds to phosphorylated carboxyl-terminal domain of receptor 3) Receptor-βARR complex enters cell via endocytosis 4) In endocytic vesicle, arrestin dissociates; receptor is dephosphorylated and returned to cell surface

Describe the 3 steps utilized by Ion Channels for selectivity of K+ over Na+

1) entries to channel have anions (to attract K+) 2) ion path narrows (selectivity filter) and dehydrates the ion, forcing it to release H2O from its "hydration sphere" 3) carbonyl oxygens inside filter form cage that fits K+ precisely and replace H2O of hydration sphere; Na+ is too small

What is an action potential? What is the flow of transfer? What are the 3 voltage gated ion channels that are essential to this? Briefly describe their functions and resting Vm's.

AP = travelling wave of electrical excitation -carried from neuron cell body through an elongated cytoplasmic extension (AXON), which triggers release of neurotransmitters at the synapse, carrying signal to next cell in circuit ION-CHANNELS: 1) Na+ Channels (ψ = +55) = depolarization 2) K+ Channels (ψ = -75) = repolarization 3) Ca2+ Channels = trigger release of aceylcholine to send message on

What is the primary type of gradient found across permeable membranes? What are its 2 composing parts?

ELECTROCHEMICAL GRADIENT 1) Chemical Gradient = difference in solute concentration across a membrane 2) Electric Gradient = difference in charge across a membrane measured by Membrane Potential (Vm) -opposes ion movements that ↑ Vm -favors ion movements that ↓ Vm

What are the 2 types of lipids and the 5 classes?

ENERGY 1) Free Fatty Acid 2) Triacylglycerol STRUCTURAL 3) Glycerophospholipids 4) Sphingolipids 5) Others

What does it mean for a cell to be "excitable"? What does the excitability of cells depend on? Give an example

Excitable = can detect an external signal, convert it to electric signal (by changing Vm) and pass it on via pathway of inorganic compounds (ions) -depends on gated ion channels -ex) Na+K+ ATPase & K+ leak channel

What are the 2 ordered states of membranes and when do they occur? What is the relationship between them @ physiologic temperatures (20-40° C)? What happens when sterols are present in the membrane?

Liquid-Ordered (Lo) =below melt temp (Tm) -individual phospholipids can diffuse laterally but acyl groups remain ordered Liquid-Disordered (Ld) = above Tm -acyl chains undergo much thermal motion and have no organization -membranes are in transition state between Lo & Ld CHOLESTEROL: -@ high MT, cholesterol decreases fluidity -@ low MT, cholesterol increases fluidity (inserts itself between phospholipids which increases distance between them and increases fluidity)

Give the 2 step process for Na+K+ ATPase in terms of how it helps with electrical signalling What are the 4 ions that are passively transported as a result. Describe their movement Which are depolarizing?* Which are hyperpolarizing?*** Which channel is open most of the time?

MECHANISM: 1) electrogenic; polarizes the membrane by establishing a Vm of -60mV (inside is negative relative to outside) 2) Ions tend to move down their electrochemical gradient across the polarized membrane IONS: *1) Ca2+ = outside (high) --> inside (low) *2) Na+ = outside (high) --> inside (low) ***3) K+ = inside (high) --> outside (low) 4) Cl- = inside (low) --> outside (high) -K+ channel is open most of the time because it's hyperpolarizing

Describe the 4 step mechanism of light reception in the eye.

MECHANISM: 1) lens focuses light on retina, which is composed of layers of neurons 2) primary neurons are rods (high-res/night vision) and cones (color vision) 3) rods and cones form synapses with interconnecting neurons that convey electrical signal 4) signals pass from ganglion neurons through optic nerve to brain

Give the following for the Chloride-Bicarbonate (anion) Exchanger found in erythrocytes (red blood cells): -name -protein classification -# of segments -general transporter classification -method of transport

NAME = AE Protein CLASSIFICATION = Type III Integral # OF SEGMENTS = 12 GENERAL CLASS = antiporter METHOD = facilitated diffusion

Give the following for the Glucose Transporter found in erythrocytes (red blood cells): -name -protein classification -# of segments -structure -method of transport

NAME = GLUT1 CLASSIFICATION = type III Integral # OF SEGMENTS = 12 STRUCTURE = helices are amphipathic (AAs separated by polarity) which produces a channel lined with polar residues -polar AAs = BLUE -nonpolar AAs = YELLOW METHOD = facilitated diffusion, which is similar to an enzyme reaction

We know that IRS-1 is activated to bind GRB-2 (an adaptor protein; what other protein can IRS-1 bind to? What is its function? What are the 5 steps of its mechanism?

PI-3 KINASE = transmission of signal for activation of glycogen synthase by insulin MECHANISM: 1) Activated IRS-1 activates PI-3K by binding to its SH2 domain; PI-3K converts PIP-2 to PIP-3 (both are membrane lipids) 2) Conversion causes phosphorylation of PKB (protein kinase B), which causes PKB to phosphorylate GSK-3 on a Ser residue, which INACTIVATES IT 3) Inactive GSK-3 can't convert Glycogen Synthase to its inactive form, so GS remains active 4) Synthesis of glycogen from glucose is accelerated 5) PKB stimulates movement of GLUT4 from internal membrane vesicles to plasma membrane, increasing uptake of glucose

What is a protein kinase? What type of molecule do they affect? What is the standard reaction scheme for a reaction involving a protein kinase? (starting vs ending) What is the reverse reaction?

PROTEIN KINASE = enzyme that phosphorylates molecules (adds phosphate group to them) -phosphorylates OH group of Ser, Thr & Tyr

SKIP

PURPOSE = analyzing AA sequences to predict membrane protein secondary structure METHOD = moving AA side chain from membrane protein from hydrophobic solvent into H2O and measuring ΔG (free energy) DEFINITION = free energy of transfer, which correlates with polarity of AA to tell us if it's hydrophobic or hydrophilic RESULTS = for successive windows of sequence, hydropathy index (ΔG values) are calculated and plotted vs. residue number

Define the following: -rhodopsin (structure/type of protein/function) -opsin -11-cis-retinal (Chromophore) -Transducin Describe the 4 steps that occur when rhodopsin absorbs light

RHODOPSIN = GPCR that acts as photoreceptor and is where visual transduction begins OPSIN = protein component of rhodopsin 11-CIS-RETINAL = light absorbing pigment; covalently attaches to opsin TRANSDUCIN = trimeric G-protein with 3 subunits: Tα, Tβ & Tγ MECHANISM: 1) absorbing photon causes 11-cis-retinal conversion to all-trans-retinal, which causes conformational change of Rhodopsin 2) Rhodopsin interacts with Transducin which catalyzes replacement of bound GDP to GTP 3) Transducin dissociates to Tβγ & Tα; Tα-GTP carries signal to cGMP phosphodiesterase 4) phosphodiesterase converts c-GMP to 5'-GMP, lowering [c-GMP] in order to keep cation channels open, causing DEPOLARIZATION of the cell (and change in Vm)

What is the Receptor Tyrosine Kinase (RTK)? Describe the general structure of RTKs relating to domain placement. Define the following 6 types of RTK's and describe the differences in structural domains. (star means elaborate): *1) INS-R 2) VEGF-R 3) PDGF-R 4) EGF-R 5) NGF-R 6) FGF-R What is the pattern among structural differences?

RTK = family of plasma membrane receptors with intrinsic protein kinase activity (phosphorylates Tyr residues on target proteins) STRUCTURE: -Outside = ligand binding domain -Inside = tyrosine kinase domain 1) INS-R = insulin 2) VEGF-R = vascular epidermal growth 3) PDGF-R = platelet derived growth 4) EGF-R = epidermal growth 5) NGF-R = nerve growth 6) FGF-R = fibroblast growth -all LIGAND BINDING DOMAINS are unique; not so for tyrosine kinase domains

What is Membrane Protein Topology? How is it done? What can be predicted about the secondary structure of a membrane protein?

Topology = analysis of primary AA sequence of membrane proteins to predict its 3D form (done "in silico") -done using enzymes that cleave in the extracellular environment (like trypsin) and can't cross membranes -peptide chain surrounded by lipiids will tend to form α-helix or β-sheet in order to maximize intrachain H-bonding (nonpolar side chains also maximize hydrophobic interactions and thereby maximize helix stability)

What is the 6 step mechanism by which an Action Potential is carried along the chain of neurons? What is the state of the neuron prior?

[Initially, neuron is polarized (inside negative) due to activity of Na+K+ ATPase, which pumps out 3 Na+ for every 2 K+ it pumps in] 1) AP is moved along axon away from cell body causing Na+ channels to open, further triggered by subsequent depolarization 2) K+ channels open in response to Na+ and repolarize the cell; refractory period ensures that unidirectional wave of depolarization (AP) reached end of axon 3) when depolarization hits axon tip, Ca2+ channels open causing influx of Ca2+ 4) increasing [Ca2+] causes exocytic release of acetylcholine into synaptic cleft 5) aceylcholine binds to receptor on postsynaptic neuron and its ligand-gated ion channel opens 6) extracellular Na2+ and Ca2+ enter through this channel and depolarize the postsynaptic cell, causing Na+ channels to open and cycle repeats