Baines

Question 1

Methods for lateral movement

Answer 1

Fused human and mouse GFP
Heterokaryons
Fused by polyethylene glycol or Sendai virus
Rapid mixing of dyes

Question 2

Rotational movement methods

Answer 2

Loss of fluorescence polarisation
Eosin maleimide reacts with AE1
Suggests a fast rotating and slow rotating population
Slow maybe linked to cytoskeleton

Question 3

Flip flop movement methods

Answer 3

Membrane impermeable enzymatic probes

Side selective membrane bleaching

Question 4

Freeze fracture of synapse and paranodes

Answer 4

RBCs show random distribution of IMPa
Other cells have adhesomes where IMPs near synapse
Overcomes free diffusion
Paranodes show high concentrations of ion channels

Question 5

Mitochondrial IMPS

Answer 5

Subjected to electric field before freezing

Normally random, but seen at one end

Question 6

Lateral diffusion after FRAP

Answer 6

Outer leaflet labelled
Bleach a small area
Lipid diffuse into bleached area restoring fluorescence
Diffusion coefficient can be determined
Integrins recovery is slow as bound to ECM

Question 7

Single particle tracking

Answer 7

Used to measure lateral diffusion
Colloidal gold attached to Integrins, becomes stuck on matrix
Gold particles are corralled by the cytoskeleton
Gold-DOPE

Question 8

Diffusion indexes

Answer 8

X10,000 difference between ACH and bacteriorhodopsin

Aggregation into purple patches stops diffusion

Question 9

Hydrophobicity scales

Answer 9

Kyle Doolittle
Hopp-woods- potential antigenic sites of proteins. Rich in charged residues, so gives a hydrophilic index. Window size 6

False negatives and positives
Does not show the 7 TMs of GPCRs

More accurate is to use a hidden Markov model which is a statistical representation

Question 10

Synthesis of class I proteins

Answer 10

N terminal signal sequence binds to a SRP
This SRP then binds to an SRP receptor before the protein is fed through the translocon
When both termini are on the same side it’s type I
N terminal signal sequence is cleaved by a signal peptidase

Will have the N in the lumen. Positive charge in near C

Question 11

Synthesis of Class II proteins

Answer 11

Hydrophobic transmembrane sequence
Due to charges which flank the TM
Positive charges always face towards the cytoplasm not lumen
So if there is a positive next to the C, then type I
Positive next to N is type 2.

When the charges are swapped, this converts 2 to 1
But without swapping, the default is a type 2 with N into cytoplasm
Signal anchor will always insert in type 2 unless charges are switched
Type 2 will have the N terminus facing inwards
Mutations in the signal sequence don’t make a difference
Default in N in

With a signal sequence, N will always be in cytoplasm

Question 12

Definition of channel

Answer 12

Approach free limits of diffusion
Not saturable
Direction by charge and gradient
Open and closing of tube

Question 13

Definition of carriers

Answer 13

Stereo selective
Below free diffusion
Outward and inward facing
50 families

Question 14

Aquaporin

Answer 14

6 TM helices and 2 half, 30 tilt with NPA loop
Cytoplasmic termini
Pore in tetrameric centre
His180 Arg195 make size and repel H3O
Water enters oxygen last but is switched by Positive dipoles of helix
Hg binds to C189

In tobacco plants transports CO2 shown by acidification

Question 15

Potassium channel

Answer 15

8 TM helices, two from each tetrameric
Wide end of cone to extra cellular
C=O of pore loops coordinate to ion
4 sites but two at a time
Mutual repulsion drives through channel, no large barriers
1000/1 selective, selectivity loop removes water shell
Large vestibule rehydrates the ions
Movement of helices thought to restrict pore

Question 16

Voltage gated postassium channel

Answer 16

4 TM helical extensions
4 beta subunits
S4 helix has Arg which is pulled to membrane
Depolarisation causes pull to extra cellular which opens channel

Question 17

The M2 proton channel

Answer 17

Acidification of endosome release virus from receptor and detach RNA
Inhibited by amantadine
Homotetramer
4 Trps block channel, coordinate with His
As pH increases, Trp points downwards instead of across and opens channel

Two possible mechanisms-
His passes protons to water
Proton hopping of water

Question 18

NA/k ATPase

Answer 18

3na out, 2 k in
Needed to maintain conc gradient and action of secondary transporters
A, b, y subunits
Beta is a chaperone and type 2
Gamma regulated by PKA and PKC by adrenaline , type 1
Alpha has Asp phosphorylation. Binds cholesterol to activate only at membrane

3 domains: nucleotide, actuator, phosphorylation

Question 19

Drugs to affect NA/K ATPase

Answer 19

Ouabain- glycosides which inhibits, mimics cholesterol. Blocks NA leaving.
Digoxin- increases cytoplasmic NA, less NCX activity which increase Ca conc to give stronger contraction

Question 20

Secondary transporters

Answer 20

Sodium always flows out to in, so after first pumping it out the reentry can be paired with another transporter

Symporters
Glucose (creates internal x30,000), nucleosides

Antiporters
Extrusion of acid (NHE) or calcium (NCX)
Action potential opens Ca in membrane and SR
Moves troponin allowing contraction
Ca pumped back to SR using ATP or exchanged for NA entry

Question 21

Secondary active transporters in kidney

Answer 21

SLC, KCC4, NKCC2 (NaKcl)
KCC3 in the PT, NCC and KCC4 in cortex and NKCC2 in outer medulla

Transporters of SLC12A are electro chemically neutral and regulate ion balance. Diuretic targets

Question 22

SLC21 transporters in kidney

Answer 22

NKCC2 brings in NA, 2Cl, K
ROMK pumps K back out
NA is pumped back into blood by exchange for K
Cl into blood by CLCK

NCC brings in NA and Cl
TRPV5 brings in Ca
NA/K exchanger
NCX1 brings NA from blood exchanging for Ca
Ca can also be returned to blood by the H/Ca exchanger (PMCA1b)

Question 23

Actions of diuretics

Answer 23

Sodium has a hydration she’ll
Acts on NA transporters to prevent water reabsroption
Furosemide- NA/KCl
Thiazides- NaCl

Question 24

Transporters in homeostasis

Answer 24

Glycolysis makes pyruvate and H before kerbs making 6H
MCT removes lactate and H
AE removes HCO3-
NHE removes H for NA
NA is exchanged for K by NKA

Question 25

NHE1 and metastasis

Answer 25

Large ATP at leading edge Extrusion of acid NHE1 and lysosomes are redistributed to the pseudopod tip to extrude acid and protease Focalised proteolysis of ECM and cell attachments Deteriorating environment gives positive feedback to NHE1 Mesenchymal to ameboid transition, moves through gaps in ECM

Question 26

NHE1 regulated by cofillin

Answer 26

NHE1 removes 2H+ from the asp122,cofillin,his133 complex At a low Ph His binds to PIP2 NHE1 makes alkali so loses PIP2 interaction Complex is activated for barbed end formation PIP2 -> IP3 and DAG by PLC

Question 27

Tertiary transporter

Answer 27

NA/K ATPase NA flows back in with AA1 AA1 then exchanged for AA2 Could be used to remove high amounts of AA1 to create useful exchange

Question 28

GLUT1 structure

Answer 28

``` 12 TM Lys mod by n-Hydroxy-succinimide esters -> 90% inhibition in Cys mutations Neutral bilayer interaction Few hydrophilic show complexes? Large N and O and cleft bottom shows C=O binding to sugars ``` In bacteria, transport H too to overcome energy, by mutating GLUT1 can create Glu/H symporter Eukaryotes lost ability as constant high glucose

Question 29

Beta barrel proteins

Answer 29

Humans have a low H gradient but high NA Gram negative have peri plasm and peptidoglycan Beta barrels excreted in peri plasm as hydrophilic before outer mem Only needs 7 AA to cross Tilted from 20-45 degrees Porins for transport e.g. Maltose Surface loops are antigenic sites Variations in Eyelet in antibiotic resistant bacteria Present in short-lived to inactive state delcour 1997

Question 30

Squalene hopeless cyclase

Answer 30

Each half is enzyme Substrate in ER membrane Long chain to sterol ring Two non polar plateaux

Question 31

Prostaglandin H2 synthase 1

Answer 31

Targets head group of A acid Channel links cavity to membrane binding surface Mobile loops release

Question 32

Myristoylstion | 2 examples

Answer 32

``` Myristic acid 14:0 to n terminal glycine Donor of myristoyl-CoA Proteolytic event of N terminal signal Specific 7 residues Regulated membrane association Permanent and cotranslational ``` MARKS- regulates actin in neurons. PKC phosphorylates Ser which repels membrane Recoverin- Ca sensor, ca exposes myristoyl group

Question 33

Isoprenylation | 2 examples

Answer 33

Strong with bilayer- targeting CAAX box c terminus Transfers from polyisoprene pyrophosphate precursors AAX removed, often methylated Precursor mevalonic acid, based around farnesyl and geranylgeranyl (X is leu/Phe) Lamin B Cell cycle dependent Rab/Ral A and B RabGDP is soluble and binds to GDI Recycled back to membrane where GEF activates

Question 34

Palmitoylation

Answer 34

Palmitate to Cys via palmitoyl-CoA by palmitoyltransferase Can be C, internal or N Reversed by hydrolysis protein acylthioesterases Membrane targeting Ras family TM proteins e,g, b adrenergic at internal Ankyrin Membrane fusion SNAP-25

Question 35

Hypothesis for palmitoylation

Answer 35

FarnesylTed protein samples membranes The PAT is present in target membrane which then fixes it Vehicle mediated transport moves it to other membranes where there are protein acylthioesterases Palmitoylation only in Golgi Makes sure that it is only present in PM

Question 36

Example of palmitoylation

Answer 36

Targetin regulation and kinetic trapping of eNOS Palmitoylation for eNOS in caveolae Down regulate by endocytosis Phosphorylation of myristoyl removes from PM De palmitoylation removes from caveolae

Question 37

Disease linked to PAT

Answer 37

ZdHHc9 is a PAT, found in complex with GCP16 in the Golgi Modifies NRAS and HRAS Upstream of MAPK which regulate transcription for division By changing 181C and 184C HRAS remains in Golgi Both lead to non specifically localised on all endomembranes Development of nervous system needs synapse maturation NMDA (Glu) -> HRAS and NRAS -> AMPA recruitment for long term signalling (exocytosis of AMPA receptors) Also migration of AMPA on dendrite surface

Question 38

Experiments for determining greasy feet | 4

Answer 38

PLC or PLD cleave at phosphate Nitrous acid cleaves between glucosamine and inositol ring releasing protein-glycol and phosphatidyinositol C terminal peptide hydrophobic in tryptic digest, will be in detergent or hydrophobic phase Acid hydrolysis reveals ethanol amine

Question 39

Biosynthesis of GPI anchors

Answer 39

Preformed anchor in ER membrane N of ethanol amine is joined to sugar C-terminal TM of protein is cleaved and added to anchor Two peaks on a hydrophobicity plot- one for signal sequence and one for c-terminal TM. Type 1 protein

Question 40

GPI anchor disease

Answer 40

``` Paroxysmal nocturnal haemoglobinuria Mutation in single haemopoetic stem cell Haemolytic anaemia Improper expression of CD55 and CD59 Mutation in PIG-A, X linked, which transfers GlcNAc to PI Higher freq due to X inactivation ```

Question 41

Types of singer proteins

Answer 41

1- N is intracellular 2 Signal anchor and c terminal anchor Poly topic

Question 42

The 3 classes of peripheral proteins | Steps of association

Answer 42

I and H penetrate the hydrocarbon core S is only interfacial region Initial membrane adsorption due to electrostatic interactions and diffusion Could be triggered by Ca switch Membrane penetration or binding to lipid For many phosphoinositide binding proteins, both occurs

Question 43

Classes of lipids

Answer 43

Tricyglycerols Phospholipids- glyceroPL and sphingoPL Glycolipids- Sphingolipids and galactolipids Ester linkage or amide linkage Phospholipids- phosphodiester Glycolipids- glycosidic linkage

Question 44

Glycerophospholipids

Answer 44

``` Derivatives of phosphatidic acid Polar alcohol can be: P-4,5BP (negative) Phosphoserine (neutral) P-choline or P-ethanol amine (positive) ```

Question 45

Phospholipase a

Answer 45

A1- first fatty acid carbon A2- second fatty acid carbon. C- cleaves between the oxygen and the phosphate D- cleaves between phosphate and head group

Question 46

Sphingolipids

Answer 46

Amino groups at C2 has amide linkage to saturated fa Ceramide is parent compound Phosphocholine -> sphingomyelin Gangliosides have very complex oligosaccharides Carrying antigens as well as barriers

Question 47

Melting points of lipids by DSC

Answer 47

Input of constant energy raises temperature At 30 degrees more temp is needed to induce a change Lipids absorb energy as they undergo structural changes Large input is the energy needed for lipid tails to melt Bending of membrane in the ripple phase

Question 48

Asymmetry in the membrane

Answer 48

Unsaturated more inward facing In apoptosis, phosphatidyserine goes to extra cellular face Sugars face outwards and create the glycolax layer Outer layer- phosphatidycholine, sphingomyelin Inner- Pserine, all PI lipids

Question 49

X-Ray diffraction of the bilayers

Answer 49

High densities of head groups Can determine where the cholesterol is by comparing to cholesterol lacking membrane In myelin produced by oligodendrocytes, cholesterol is not symmetrical

Question 50

The raft hypothesis

Answer 50

Different ratios of components makes Ld, Lo and So regions | Phase separation of phosphatidycholine and sphingomyelin upon cholesterol addition

Question 51

Calveolin

Answer 51

Peripheral Monomers has 3x acyl groups by palmitoylation and myristoylation Polymerises when contacts cholesterol Central hydrophobic domain

Question 52

Raft function in T cell activation

Answer 52

CD45 dephosphorylates the tyrosine kinase LCK Can also act on LCK substrates such as ITAMs Ligation of TCRs pulls rafts together, by excluding CD45 this activates LCK Allows signalling

Question 53

Detergent resistant membranes

Answer 53

GPI anchors became insoluble Associated via acyl chains with phospholipid acyl chains such as SM and PI. Cholesterol also stabilises Tightly packed bilayer Low density Cyclodextrans which remove cholesterol disrupts rafts But detergent can cause artifacts

Question 54

Proteins to change lipid bilayers

Answer 54

Flippases - change aminophospholipids from outer to inner Need ATP, p atpases Floppases- from inner to outer need ATP , ABC transporters Scramblases- no ATP, activated by calcium. During cell death Ca increases, so phosphatidylserine exposed on extra cellular

Question 55

Synthesis of dolichol phosphate

Answer 55

``` Addition of GlacNAc by UDP Mannose addition Translocation (flipase) Transfer to Asn residue Protein in ER lumen ```

Question 56

Lipid linked diseases

Answer 56

Tay-Sachs- underdeveloped, paralysis, blind. Abnormal ganglioside deposits in the lysosomes Affects the enzyme hexoseminidase A which removes GalNac Niemann-pick- defect in sphingomyelinase. Prevents the breakdown of sphingomyelin into Ceramide and phosphcholine

Question 57

Lipids as signalling molecules

Answer 57

PIP2 -> DAG -> CALDAG-GEFI -> Rap1 -> RIAM PIP3 is a marker for recycling endosomes in epithelial cells PIP2 ->PIP3 actives PKB which inhibits GSK This allows glycogen synthase to be activated Dynamin PH domain binds to PIP2 whereas a GED domain is involved in the oligomers satin and regulation of GTPase activity

Question 58

Tetraspanins

Answer 58

Large family with 4 TM segments 10^4 per cell except from red cells Aggregate with other proteins in membrane CD151 is a glycoprotein which controls neurite outgrowth. Associates with a3b1 Integrins X linked mental retardation Huntingdons, fragile X and myotonic dystrophy

Question 59

The CO2 metabolon

Answer 59

``` AQP1 RhAG CD47 Glycophorin B 4.2 AE1 Ankyrin Carbonic anhydrase II ``` Decrease in pH causes oxygen release

Question 60

Hereditary spherocytosis

Answer 60

``` 1/5000 Small rounder and more fragile Trapped in the spleen High levels of production in bone marrow Aplastic crisis Increased numbers of reticulocytes Often treated by removing spleen to prevent breakdown Young kids at risk of sepsis with pneumococcal bacteria ``` Dominant: b Spectrin W202R, ank V4631, ae1 P327R, Also rh null and 4.2 null Recessive- heterozygosity

Question 61

Hereditary elliptocytosis | Pyropoikilocytosis

Answer 61

Causes elliptical shaped cells Either 4.1 null or inability for Spectrin tetrameric Severity upon dimer:tetramer B Spectrin L2025R, a Spectrin K48R V31A, protein 4.1 M1R Sensitivity to to heat

Question 62

Does spectrin bind to the membrane?

Answer 62

Radioactive labelled Bind inside out vesicle Binds to Ankyrin (palmitoylated) Ankyrin binds AE1

Question 63

Regulation of spectrin binding

Answer 63

Spectrin and actin coextract 4.1 and adducin also present Adducin binds to + end

Question 64

The AE1 complex

Answer 64

``` Attached through Ankyrin to Spectrin Tetrameric Protein 4.2 GPA, rh and rhag bind to AE1 CD47 and LW associate ```

Question 65

Protein 4.1 complex

Answer 65

``` Membrane skeletal junctions Spectrin, f actin, 4.1 Actin binding proteins Ternary interaction with p55 and GPC Rh, kell and XK Dimer ae1 ```

Question 66

Sickle cell and haemoglobin

Answer 66

Deoxy HbS forms contacts between molecules and polymerises Instead of a charged residue, can dock into a hydrophobic pocket (val6) When water levels, there is more aggregation

Question 67

Sickle cells and oxidation

Answer 67

Haeme and free iron leak leading to oxidation of GSH This means that GSH cannot be oxidised to GSSG to protect Oxidation activates the psickle channel This allows calcium entry KCl is lost via the gardos channel Prevents colloidoosmotic lysis In normal cells, de oxygenation deactivates the gardos channel Senicapoc inhibits the Gardos channel and stops dehydration

Question 68

C284 and C373 in actin

Answer 68

``` Disulphide bond between C284 and C373 Because of glutathionation of Cys residues Cytoskeleton locks Even when oxygenated still appear sickle Prenyl-actin used as a probe ```

Question 69

N-acetyl cysteine

Answer 69

Potential to reduce oxidative stress It is the rate limiting substance for production of GSH production It enters cell and is converted to cysteine Increasing GSH prevents oxidative damage

Question 70

Beta Spectrin mutations in c.elegans

Answer 70

SR lost Muscles tear In heart cells, the SR is lined with Spectrin. If it can't resist mechanical stress this leads to heart malfunction

Question 71

Ankyrin B knock out mice

Answer 71

Loss of function mutation E1425G Disrupts organisation of the NA pump, NA/Ca exchanger which reduces the targeting of these proteins to the tranverse tubules This causes altering of Ca signalling

Question 72

What is the effect on Ankyrin B loss in cardiomyocytes?

Answer 72

Gradient cannot be created by NA/k ATPase Gradient is coupled to extrusion of Ca by NCX Ankyrin stops reinternalisstion Cardiomyocytes cannot reset after calcium release Irregular heartbeat and sudden death

Question 73

Spectrin and evolution

Answer 73

Binding partners integrated cells into tissues Two rounds of whole gene duplication 4 beta spectrin said, but one alpha In mammals alpha was duplicated and specialised for red cells (a1)

Question 74

Isoprenylation and heart disease

Answer 74

Statins increase lifespan Decreases specific protein prenylstion Explains the non cholesterol related affects of statins 2012 clinical trial to inhibit prenylstion to treat Hutchinson Gilford progeria syndrome which causes accelerated atherosclerosis

Question 75

Lipid linked diseases 2

Answer 75

B-galactosidase: generalised gangliosidosis Hexosaminidase A: Tay Sachs, GlcNAc removal Gauchers: glucocerebrosidase, removes Glc from Glc-Ceramide Niemann pick: sphingomyelinase Fabrys: a-galactosidase A Sandhoffs: hexosaminidase A and B