Midterm one Flashcards

Question

What is cardiolipin?

Answer 1

Has been shown to have a high affinity for curved membrane regions (ex. inner mitochondrial membrane) Present in mitochondria, smaller more compact head group with more range on tail, helps with curvature Four fatty acid chains, two PA lipids coming off glyceriol (diphosphatidylglycerol) - 18C fatty acids with 2 unsaturated bonds

Answer 2

Lipids have polar and non-polar components (overall polar though) Can form up to four hydrogen bonds Responsible for the formation of lipid related structure In water you get H bond lattices of water, upon addition of hydrophobic molecules, some of the bonds will break

Answer 3

-Decreased entropy -Will drive hydrophobic molecules together to diminish contact with water stability of H bonded water network make it difficult to dissolve hydrophobic compounds Introduction of lipids leads to decreased stability and a decrease in entropy OVERALL happens because: Large positive free energy is not favorable Enthalpy change of taking a hydrocarbon and putting it into water is very small Large negative change in entropy dominates the change in free energy We overcome this by aggregating the non-polar molecules together

Answer 4

Surfactant has a hydrophilic and hydrophobic mobility, partition of water oil interface Depending on specific surfactant it can lead to different divisions OR at high concentrations you get c which gets you more of mixing of phases; comes back to concentration of components ex lipids of surfactant

Answer 5

Critical micelle concentration: the concentration at which the lipid molecules aggregate and form micelles Depends on the chemical structure of CHAIN LENGTH AND HEAD GROUP

Answer 6

In vesicle has aq on inside and outside (like a cell sued to trap hydrophilic particles in vesicles) In micelles the stuff inside is hydrophobic tho Soap will form micelle's structure when you start to wash hands, agitate hydrophobic stuff ex dirt Agitate micelles which start to trap them inside micelles On outside of micelle is hydrophilic head groups which is easily washed away with water

Answer 7

Vitamin E is a common bimolecular marker found in bracheoli which is related to lung stress with vaping They wash the respiratory system, and the unwanted debris come out Vitamin E is one of the many dilutants of different chemicals, used in vape flavoring When vitamin e you get a much softer membrane which means it cannot withstand certain pressure with constant expansion/exhale leading to the collapse which leads to alveoli death Initially people think vitamin E is good for you but it's bad here. In vaping industry, they base safety regulations of FDA which tests eating/consumption NOT investigating inhaled which is why it passed

Answer 8

-Shape parameters: ratio of head size to tail size will give certain structure ex. More planar or curved bilayers -Curves can come in the form of folds in bilayer (helps with rounding affect and some processes ex. Endo and exocytosis, membranes go in and out) -Shape parameter influence shape and affects function.

Answer 9

Form planar regions where you can incorporate lysolipids (phospholipids with a cleaved tail so head tail is bigger than tail region to form the curved part. There is also certain charges which will help with curvatures. Gel like = solid, L alpha is more fluid Previously thought of saturated vs unsaturated fatty acid BUT here is the melting point that determines what phase; at room temp they are almost always in liquid phase but if more saturated or below temp then rigid phase

Answer 10

Depending on size parameter you will get positive or negative curvature There is a bar domain which helps stabilize curvature; proteins form dimers that is stabilized upon binding to lipid membrane, -Binding via electrostatic interaction because membrane is negatively charged (strong binding = more curve) -Bind to more readily curved liposomes therefore some membranes have a higher affinitive for curvature since there is more electrostatic interaction opportunities Certian residues stabilize curvature There is also specific lipid domains that help, like PI lipids, which are used for signaling and bind to specific protein with the domains close to membrane which stabilize the curve process

Answer 11

Cholesterol has affinity the Ld phase (which changes La to Ld) -Ld is more disorder -Lo is more ordered -Lb is more solid General effect is to inhibit Lbeta but result in a less fluid and more ordered structure than L alpha -Degree of cholesterol will determine of Lo or Ld phase, once you bypass the lipid threshold for cholesterol you will enter the Lo phase -Never just all one type, ex. If Ld phase there will be small amount of Lbeta still, it will just be negligible

Answer 12

1. Fluorecence Microscopy 2. differential scanning calorimetry (DSC) 3. Scattering experiments (look at membrane and phase behavior through neutrons)

Answer 13

-Can use flourescently labelled lipids to look at lipid phases; visualize so most people can understand -Pros Easy to obtain Quick to visualize, see different phases, people understand well Nondestructive probe, doesn't damage system -Cons Exogenous (bulky) probe –-> probes that you attach to lipids are bulky which can cause the lipids to deviate from their natural structure Photobleaching --> fluorescent probe has the possibility of being exposed to light which will then degrade. This causes it to be less accurate Auto fluorecent cells are cells that have innate florescent behavior so if you are using fluorescent to quantity a certain behavior in the cell it can throw off numbers

Answer 14

Use two cells, one sample and one reference, can be done with solids but when using lipids normally the one of interest is in an aqueous solution and the reference will be in water Add heat to cells slowly at the same rate Once you get close to melting point you are breaking the interactions of gel phase Need energy to break the bonds, there is difference in heat transfer because as system absorbs heat (to break the bonds) the cell must compensate by adding heat Therefore, in water nothing will happen, no absorb or release of heat However there needs to be energy to go from gel to disordered aq phase. On graph the area under peak is enthalpy related to that specific event In some cases, there is a lipid ripple phase (intermediate/ pre transition peak)), the intermolecular forces start to breakdown which may cause a small hump before dissolving into full phase Peak is full melting phase

Answer 15

1. Data collection: investigation of samples with X-ray or neutron scattering technique 2. Data reduction: raw data corrected by background or instrumental effects, normalized etc. 3. Modeling: modeling structure (ex. Bilayer) can look at electron or neutron density distribution associated with a structure 4. Fitting data adjusts parameters chosen by model to best match data 5. Parameter extraction: fitted model will be based on preset parameters (can lock value) 6. Probability profiles: information of electron or neutron density 7. Analysis and interpretation: parameters and profiles can be used to gain insight on structure or function 8. Software tools: large number of tools for analyzing data available

Answer 16

Make sample based on area of interest Background reduction you want a baseline reading to subtract from all the data Model the reduced data. Structural model to help depict data Match data to theories Based on scattering pattern to pick theory and then get parameters based on it You can also get profiles which helps with depicting what structural component you are looking at, differ between x rays and neutrons because they highlight different aspects of membrane (x rays interact with electrons in sample so more e rich areas lit up, so in a phospholipid the phosphate (head) is more lit up; look at the neutrons of sample)

Answer 17

1. X ray scattering 2. Neutron scattering 3. Mass spectrometry: has been used for lateral organization but not transverse, and characterization --> separate all the lipids of the bilayer and tun it through process --> just look at single area/ specific structure to get a lipid profile then compare against literature 4. Solid state NMR: for info related hydrocarbons, used to confirm lipid asymmetry 5. CryoTEM; one of more accessible approaches, quick measurements, get a picture that is easy to interpret; electrical microscopy angel, visualizing technique, flash freeze sample at specific time, cannot look at dynamic process 6. Cross section of bilayer

Answer 18

Beak of neutron/xray --> hits sample and angle is detected --> machine has different techniques based on how machine was built Can give insight on structural parameters of vesicle systems 1. SAX: nanoscale resolution (1-100nm); structural properties Some structural parameters measured with the visualization of SAX -Bilayer thickness, head-to-head thickness, area per lipid, lipid volume, water thickness, and hydrophobic thickness 2. WAXS: atomic resolution 0.1-1nm); atomic details WAX (wide resolution) the farther your sample detector is the more scattering angle you can get so you need bigger detector to capture all the incoming signals If you use wide angle than refraction can give more detail Data gives intensity curve based on where data hits; means scattering event occurs more than others

Answer 19

-Pros 1 Nanometer scale resolution 2 Quantitative information 3 Dynamics studies 4 Low sample requirements Cons 1 Average structural description 2 Radiation damage 3 Instrument access 4 Data interpretation

Answer 20

Neutron will interact with the sample and scatter at an angle at the detector ; looks at scattering intensity -Can look at : 1. Size and structure 2. Domain formation 3.Monitoring asymmetry -Process 1 Neutron source 2 Hit the scanner 3 Depending on how it scatters you have a circular map giving intensity (how many points hit that specific q value) vs q value (where it refracts)

Answer 21

Pros -Sensitive to H -Contrast variation methods -Dynamic studies -Less damaging than x rays Cons -Lower flux compared to x rays -Expensive and hard to get access -More complex analysis -Large amount of samples required

Answer 22

1. small angle X-ray scattering (SAS) 2. small angle neutron scattering (SANS) 3.cyro electron microscopy (make note of name, pro/con, and what it says)

Answer 23

The methods by which the neutron scattering intensity from a multicomponent complex can be separated into that from the components through the use of hydrogen- deuterium substitution in the complex or solvent The difference in scattering length density between materials is the “contrast”

Answer 24

Allows for the visualization at the near atomic level Vesicles need to be frozen and embeded specialized grid Programs can be used to analyze image to obtain structural data

Answer 25

Pros -Preservation of sample -Versatile (size range) -Minimal radiation Cons -Cost/specialization -Computationally intensive -Non moving system

Answer 26

-Single and Nicolson; the fluid mosaic model of the structure of cell membranes --> systems/bilayers that have variety of different lipids in systems which give memebrane some function which might lead to downstream signaling -Kai Simons Determination of lipid rafts Hypothesized a specific interaction between glycosphingolipids and apical proteins Luminal leaflet has to be concentrated by sphingolipids for vesicle transport Built on the idea of lipids domains forming on cellular membranes Saw that different protiens were packaged in different areas of the cell; differenjt lipids sent to different areas of the membrane

Answer 27

Can get bigger and smaller in size; transient; smaller and always dynamic; appearing and disapearing depending on functional needs

Answer 28

1. GPI anchored protiens GPI- GlycosylPhosphatidylInositol Attached to the outer leaflet of the cell membrane Can function in cell adhesion, as enzyme, receptors, proteases, or structural purposes 2. Src Kinase (ex. C-Src) Typically found on inner leaflet Dynamic association Dysregulation has been linked to cancer progression (hyperactivated) Can function in signalling, adhesion, migration, survival and angiogenesis Lipid raft targeting has been a focus of cancer therapeutics

Answer 29

-One problem with models is that you lack behavior of an actual biological sample -Distribution of phospholipids in bilayers Has to do with the composition of membranes Overall lipid composition on inner and outter leaflets but there is also asymmetry in inner and outter

Answer 30

1.Phosphoglycerides: synthesized on cytosolic face of ER (major site of lipid syntesis -Can freely exchange leaflet in ER -Not the case in the PM 2.Golgi: can be involved in lipid modification and processing 3.ER does not have sig asymmetry but plasma membrane and golgi have the P4 translocate enzyme in turns of lipases and flopases 4. Protiens 5. Vertical synthesis -SM: synthesized on luminal face of golgi --> outside of PM -Ceramide made in ER is major building block of sphingolipids -studying occurs with some sort of probe; involves ability to go from one leaflet to another --> hydrophobic/philic proeprties may become an issue -Ideally probe free system; neutrons system is less invasvie

Answer 31

Flippases: movement of lipids from outer to inner leaflet (require energy) Floppases: movement of lipids from inner to outer leaflet (require energy) Scramblases: bidirectional movement causing randomness

Answer 32

Membrane phase influences flip flop Membrane geometry influences flip flop Methods to look at flipflop 1.NMR 2.ESR (bulky probe, may affect transfers ability of lipid) 3.Neutron scattering 4.MD (molecular dynamics, want to see in real life) 5.SFVS (some frequency vibration spectroscopy) (lots of bilayers fixed to a certain plate, free floating vesicle in a certian solution) 6. mediated flip flop

Answer 33

Looking at h contrast variation Idea of protien or dna match to solvent Monitor change in neutron scattering intensity over the function of time Use protiated and deuterated lipids will give good contrast Symmetric vs asymmetric will have different SLD and therefore different scattering intensity curve Do as a function of time Start with asymmetric system but then symmetric will be your average mix scrammled LEB? Roughly 50/50 eq state (maybe like 60/40 depedning on curvature, maybe more lipids on outside) Average SLD of two lipids and match sysetm to it, solvent system will be matched to the symmetric theroretical scrammbling whoich is why asymetric graph line levels

Answer 34

Idea of using probes Bigger probes may cause deformaties in bilayer which will affect the halflife Protiens may have harder time going through transverse membrane (due to hydrophobic/philic) and will want to go through probe because easier --> probe leads acess from inner to outter leaflet Can get rid of asymetry; laterally diffusing over transverse

Answer 35

Goes back to lipid asymetry and protien Cytosol and exocytosis side Negative charges can attract positive amino acids which helps with structures certian transmembrane proteins So asymmtric distribution of lipids causes different distribution of charges which can affect the function

Answer 36

1.Peripheral Can be removed or added as needed 2.Lipid anchored Strong lipid modification 3.Integral membrane proteins Also known as “intrinsic” --> spans membrane

Answer 37

-Associate through strong hydrophobic interactions and the interactions exposed to the cytosol is hydrophilic Can be isolated by: 1.Detergents – dissolve sections of the transmembrane with some phospholipid attached (maintain function hopefully) 2.Chaotropic agents (don't care much about preserving function, fully denature, but you can gain insight on a.a seq) 3.Denaturing conditions Are amphiphiles 1.Fatty acid interacts with r group 2.Hydrophobic surface residues in core 3.Polar residues on extended portions Demonstrated by surface labelling Labelled protein domain with agents that cannot penetrate the membrane

Answer 38

** they will cross 0 line on the hydropathy plot 1.glycophorin A 2.Bacteriorhodopsin 3.OmpX and PapC usher 4.Ion channels 5.Resting K+ channels 6. Voltage gates channels

Answer 39

-transmembrane protein -Located on red blood cells Functions: 1.Membrane integrity 2.Cell to cell interactions 3.Lipid raft association 4.Blood type (MNS) --> carries blood group antigens

Answer 40

Online tool that can be used to predict transmembrane regions based on residue seq Uses a hydrophobicity sale as a predictive tool Get sequence. Above 0 is hydrophobic and below is hydrophilic This will tell you if protein spans membrane. Once you gain region that passes threshold/0 then you have confidence it spans membrane. Whever ABOVE 0 is a transmembrane region

Answer 41

Bacteriorhodopsin; a helices, varies in function -transmembrane protein Residues must organize exposing hydrophobic regions to fatty acids hiding polar backbone; alpha helices, beta sheets Bacteriorhodopsin (alpha helices); 7 transmembrane helices, has a retinal chromophore, changes from cis to trans upon light absorption to activate light driven proton pump = ATP production Cis to trans allows protons to go across membrane

Answer 42

-transmembrane protein Beta barrels occur in porins = channel- forming proteins Initially seen in bacteria --> organelles Seen in cell membrane of bacteria or organelles. Forms pore on inside to transport ions or small molecules --> typically in an outer membrane to shutter small molecules through the protective outer layer --> typically just for movement

Answer 43

-transmembrane protein Relevant during resting state of a cell Allow movement of K out of cell through confirmational changes Activity regulated by voltage across the membrane Vestibule: cavity or chamber-like structure that is part of a transmembrane proteins

Answer 44

Certain molecules will have easier time than others, different proteins are required

Answer 45

Once they are in the membrane, most of function is from helices rather than extracellular components Tetramers and monomers

Answer 46

Topogenic sequence: a peptide seqence essential for a protien to properly insert and orientate in the membrane Single pass vs multipass

Answer 47

N terminus signal sequence enter translocon until reaching a stop transfer anchor sequence (STA) Typically will be hydrophobic residues to make a helix Stops translocation through the translocon and will release into the bilayer Synthesizes remaining C-terminal portion until stop codon Can move laterally between protien sub units

Answer 48

Instead of STA they will have a signaling seq that functions in bringing it to the locon and The postive charges of concentrated residues on protien affect whether the initial part will go through the translocon or if it will sequeeze around? IN TYPE TWO THEY WILL NOT GO THROUGH Orientated in the translocon with N terminal towards cytosol Believed to be mediated by positively charged residues Alongated with an internal signal anchor (SA) sequence; also binds SRP Laterally moves out and anchors into membrane Completed with C terminus being released into lumen side

Answer 49

Similar orientation to type I however does not have signal sequence Similar to type II, however terminals are opposite Signal anchor sequence is located near N terminus Have positive residues on C terminal side, help orient SA sequence

Answer 50

Idea that they are multi spanning How many spanning regions you have will say if coo or nh group will be on same or opposite sides Ex if even number then terminals will orientate on same side; alternatively there is one on outside and one on inside if odd number

Answer 51

Allows for an otherwise water soluble protein to interact strongly with membranes Allows sorting of proteins into particular membrane domains (eg rafts) Acts as a functional switch (on/off = with or without)

Answer 52

1. Prenylation 2. Myristylation 3. Thioacylation 4. Cholesterol modification 5. GPI anchoring

Answer 53

S-acylated proteins co-purify with cholesterol-rich detergent- insoluble membranes domains – Prenylation = branched = bulky – Acylation = saturated = not bulky Looked at detergent resistant membranes (DRM) from Madin- Darby canine kidey cells – Isolated and proteins were characterized * Ras – Was it or was it not found? * Src family kinases – Was it or was it not found?

Answer 54

Example of rational drug design Aim was to inhibit farnesylation of Ras, which is often mutated in overactive cancers Several canditdates showed promise in cell culture and mice but crashed in Phase III trials Reason: likely GG-ation of K-Ras and N-Ras Combination of Ftase and GGTase inhibitor is toxic -For therapetuc use; lets say ypou dont want a spoecific protien to reach the membrabn; form an inhibior that targets gernaylgeranyl --> in cases of no gg the cell can still adapt so there still might be some loalization on membrane --. If you inhibit one enzyme, other protiens may go and restore function as theyve evolved to have that behaviour

Answer 55

Localized to inner leaflet of PM * Myristoyl moiety, as well as polybasic region nearby, accounts for membrane affinity * Membrane association necessary for biological activity as substrates are membrane-associated * Phosphorylation of serines in polybasic region results in membrane dissociation * Ex) c-Src had higher kinase activity with mod

Answer 56

Prenylation- covalently attached lipid built of isoprene subunits -Typically, farnesyl (C15) and geranylgeranyl (C20) Isoprene; base unit FPP; helps it traverse the membrane; Proteins will have residue req that is prone to these modifications. For exame Farnesyltransferase and geranylgernyltransferases responsible for modification Post addition mods can be followed to help with insertion and stability Typically done at C terminal of protein

Answer 57

Linkage of FA to cysteine residue by reversible labile thioester linkage [palmitate (C16) most common] -75% * Exclusively post-translational – Enzymes are membrane associated (S-acyl transferases) * Initially hard to study due to lack of consensus sequence * Reversible -thioester linkage ----- Occurs in peripheral or transmembrane domains * Often can co-exists as dual lipid modification – such as with myristoyl

Answer 58

-Occurs in Hedgehog (Hh) family of signaling proteins involved in development * Conserved GCF tripeptide gets cleaved by auto-processing domain * Cholesterol can act as trafficking signal

Answer 59

Member of the ZDHHC family, originally named as reduced expression associated with metastasis protein (REAM) – Tumor-suppressor protein – Expression has implication in cancers * Acts as a palmitoyltransferase – Substrate? * CKAP4 – cell surface receptor for antiproliferative factor (APF) * APF leads to depalmitoylation  nuclear translocation  gene regulation

Answer 60

Morphogen will form a gradient with individual cells in terms of concentration. In response to the concentration a specific differentiation event will occur in relation to the concentration.

Midterm one Flashcards

(89 cards)