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Biology 2B03 > Module 3 - Biomembranes and cell architecture > Flashcards

Module 3 - Biomembranes and cell architecture Flashcards

1
Q

reasons to purify proteins

A

structural analysis, functional analysis, determination of amino acid sequence, development of antibodies to a protein

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2
Q

steps taken for purifying proteins

A

1) develop a good assay
2) select a protein source
3) break open cells - protein extract
4) solubilize protein
5) stabilize protein
6) fractionate
7) determine purity

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3
Q

protein assay

A
  • method of detecting for the pressure of a specific protein and estimating the concentration of that protein
  • the assay should be specific to the protein of interest by basing it upon unique characteristics
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4
Q

protein source

A
  • source must be one in which the protein of interest is easily obtainable in large amounts
  • contains high concentrations of the protein
  • low in proteins that may co-purify
  • low in proteases that may destroy the protein of interest
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5
Q

protein solubilization

A
  • can predict solubility based upon what you believe its properties are
  • protein solubility is affected by the pH of the solution, salt concentration
  • insoluble proteins are affected by detergents. the presence of detergents can increase the solubility of insoluble proteins in a solution
  • protein solubility is important to consider so we can determine gel content, separation means, etc
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6
Q

examples of soluble proteins

A

cytosolic proteins, secreted proteins

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7
Q

examples of insoluble proteins

A

transmembrane proteins, membrane-associated proteins

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8
Q

important parameters to consider in protein solubilization

A

temperature, protease inhibitors (to prevent degradation), ligands, salts, metal ions, concentration, pH

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9
Q

fractionation

A

separate proteins into different groups or fractions

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10
Q

protein fractionation

A
  • many different techniques to do this
  • each technique takes advantage of unique properties of the proteins that distinguish it from other proteins
  • must apply multiple different techniques because usually each technique targets one particular unique property
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11
Q

protein fractionalization techniques: size

A

gel electrophoresis, gel filtration chromatography, ultracentrifugation

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12
Q

protein fractionalization techniques: charge

A

ion exchange chromatography, gel electrophoresis

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13
Q

protein fractionalization techniques: polarity

A

adsorption chromatography, hydrophobic interaction chromatography

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14
Q

protein fractionalization techniques: specificity of binding

A

affinity chromatography

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15
Q

differential centrifugation

A
  • used for protein isolation
  • separates proteins by mass/density
  • process: tissue/cell extract is spun at 1 000g to obtain a pellet containing nuclei and chloroplast. the supernatant is spun at 10 000g to obtain a pellet containing mitochondria. the supernatant is then spend at 100 000g to obtain a pellet containing microsomal fractions. content remaining in supernatant is cytosol
  • can stop at any one of these sections of the process if the preferred content is present in the form of the pellets obtained
  • otherwise, this process must be continued as the preferred content is still in the supernatant
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16
Q

separation by chromatography

A
  • separated based on degree of interaction with substance within the column
  • more interaction = retarded to a greater extent
  • less interaction = retarded to a less extent
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17
Q

ion exchange chromatography

A

separates based on charge. if the column has positively charged particles, the positively charged particles in the sample will elute first as they are repelled by the column. negatively charged particles will be attracted to the column and elute more slowly. the negatively charged particles can be obtained from the column by adding a salt solution that will disrupt the ionic interactions between the particles in the sample and those in the column

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18
Q

gel filtration chromatography

A
  • separates based on size
  • molecules small enough to travel into surface bead depressions are delayed and travel more slowly
  • can order beads for columns with specific cavity dimensions that match with the particles of the sample that need to be delayed
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19
Q

affinity chromatography

A
  • separates based on specificity of binding to another molecule
  • beads are covalently attached to antibody
  • antibody will only specifically associate with antigen
  • protein recognized by antibody will stay in the column due to non-covalent interactions with antibody. this is our protein of interest
  • the protein can be eluted from the column by disrupting the non-covalent interactions it has formed with the antibody
  • can use something else besides antibodies. as longs it is specific
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20
Q

SDS-PAGE electrophoresis: sample preparation

A
  • denature proteins to eliminate the effect of shape

- addition of a negatively-charged detergent that associates with all proteins to eliminate the effect of charge density

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21
Q

SDS-PAGE electrophoresis: protein separation

A
  • proteins can now be added to the gel
  • proteins will move based on molecular weight
  • large proteins will move slowly while small proteins will move quickly
  • different lanes in gel represent different fractionation processes performed to obtain desired protein
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22
Q

SDS-PAGE electrophoresis: western blot

A

immunoassay technique commonly used to separate proteins in a complex mixture and then identify the protein of interest using specific antibodies

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23
Q

specific activity

A

enzyme activity/amount of protein (mg)

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24
Q

multi-procedure/steps for purification of a hypothetical enzyme

A

1) crude cellular extract
2) precipitation with ammonium sulfate
3) ion-exchange chromatography
4) size-exclusion chromatography
5) affinity chromatography

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25
Q

unaided eye

A

100um-10m

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26
Q

light microscopy: conventional and fluorescent

A
  • uses visible light

- 100nm-1cm

27
Q

electron microscopy: scanning and transmission

A
  • beam of electron

- 0.1nm-100um

28
Q

limit of resolution (D)

A
  • the minimum distance between 2 objects in which the 2 objects can be distinguished from one another
  • if 2 objects are closer than D, we cannot use the microscope
29
Q

numerical aperture (NA)

A

a measure of the ability to gather light and resolve specimen detail

30
Q

improving resolution

A

decreasing wavelength = decreasing D = better resolution

31
Q

Nomarski (DIC) and phase contrast microscopy

A
  • they are complimentary techniques capable to producing high-contrast images of unstained, unfixed transparent specimens
  • both rely on enhancing the inherent differences in density of different regions of cells
  • live specimens can be viewed with both techniques
  • phase contrast tends to favour clear visualization of internal cellular structures while DIC provides clear images of the surfaces and edges
32
Q

immunofluorescence microscopy

A
  • use this to view specific locations of molecules within a cell
  • molecules of interest are tagged with fluorescent dyes/antibodies
  • primary antibodies are produced that recognize primary antigens
  • secondary antibody recognizes and binds to the primary antibody
  • secondary antibody is covalently attached to a fluorescent molecule call fluorophore
  • fluorophore is excited by UV light, so it emits another wave of fluorescent light, allowing us to detect the targeting protein
33
Q

Green Fluorescent Protein (GFP)

A
  • natural fluorescent protein of jellyfish
  • can be used to tag proteins in our own bodies by using recombinant DNA technology to create a gene fusion between the protein of interest and the gene coding for GFP. we then reintroduce this fused gene into our own bodies
34
Q

confocal scanning microscopy

A
  • can obtain high resolution images from fluorescently-labelled samples
  • uses lasers to excite certain thin optical layers of a cell
  • clear image is created by eliminating background fluorescence above and below layer
35
Q

deconvolution microscopy

A
  • creates clear image by using traditional fluorescence microscopy
  • fluorescence image is processed through computer to eliminate out-of-focus fluorescence that is above and below the focal plane
  • creates digital images
36
Q

transmission electron microscopy (TEM)

A
  • increases magnification by improving resolution
  • beam of electrons are directed at sectional specimen
  • D=0.1nm
  • stained, dense areas of specimen appear dark
37
Q

scanning electron microscopy (SEM)

A
  • beam of electron are directed at the surface of solid specimens
  • reveals information about surface morphology and texture
38
Q

biomembranes

A

allows for the unique creation of a molecular environment that is separate from other parts of the cell

39
Q

distinct biomembrane surfaces

A

cytosolic, exoplasmic, lumenal

40
Q

exoplasmic membrane surface

A

faces outside of the cell

41
Q

cytosolic membrane surface

A

faces inside of the cell (i.e. the cytosol of the cell)

42
Q

lumenal membrane surface

A

faces interior of organelle

43
Q

bilayer structure of biomembranes

A
  • row of polar head groups face outside and inside of the cell
  • hydrophobic tails face one another, forming a hydrophobic core
44
Q

phospholipids

A
  • basic unit of biomembranes
  • amphipathic
  • in an aqueous solution, phospholipids will spontaneously arrange themselves to form a micelle. this is not energetically favourable
  • at higher concentrations of phospholipids, their amphipathic nature allows them to spontaneously form into a bilayer. this is energetically favourable
45
Q

micelle

A

forms when a single sheet of phospholipid assembles with one layer of hydrophilic molecules and the hydrophobic core

46
Q

chemical makeup of phospholipids

A
  • diglycerides contain 2 fatty acids linked to glycerol
  • diglycerides contain charged phosphate group attached to 3rd OH group of glycerol
  • fatty acids contain hydrocarbon chain capped with carboxyl group
  • fatty acids are hydrophobic, phosphate group is hydrophilic
47
Q

why do biomembranes have distinct activities, although they are composed of the same basic structure?

A

its collection of proteins varies, resulting in varying functions for the membrane

48
Q

integral membrane protein

A
  • embedded in hydrophobic core of bilayer

- single-pass TM protein, multi-pass TM protein, beta-barrel, channels

49
Q

lipid anchored proteins

A
  • anchored in one leaflet by covalently attached lipid modifications
  • protein modifications: acylation, prenylation
  • acylation: adds lipid anchor to N-terminus
  • prenylation: adds lipid anchor to C-terminus
  • uses GPI anchor. this forms hydrophobic anchor attaching protein to the membrane
50
Q

peripheral proteins

A

attached to polar surface of membrane or indirectly by other proteins

51
Q

biomembrane fluidity

A

allows for the fusion of biomembranes and for lateral movement within the membrane

52
Q

leaflet

A
  • layer of a phospholipid membrane

- inner and outer leaflets have different characteristics

53
Q

fluid mosaic model

A
  • describes structural features of biological membranes
  • plasma membrane is described as fluid because the components, phospholipid, and membrane proteins move laterally through the membrane
54
Q

FRAP

A
  • allows for the measuring of the fluidity of the proteins of a membrane
  • membrane proteins are fluorescently labelled and therefore can be excited by UV light
  • too much UV light and the fluorophores will be bleached. they won’t fluoresce
  • fluorescence recovery
55
Q

fluorescence recovery

A

diffusion of fluorescent proteins back into bleached area. done by mobile fusion proteins

56
Q

regulating membrane fluidity: lipid composition

A

increasing order in bilayer will decrease fluidity, decreasing order in bilayer will increase fluidity

57
Q

regulating membrane fluidity: temperature

A

lower temperatures will decrease fluidity, higher temperatures will increase fluidity

58
Q

lipid raft

A
  • microdomains in membranes that are composed of the less fluid component
  • its slightly taller and higher cholesterol concentrations make the membranes with lipid rafts less fluid
  • lipid rafts as a unit are mobile within the membrane
59
Q

is there movement between leaflets?

A

it is hard to move phospholipids from one leaflet to another because their polar head group needs to pass through the hydrophobic core. this requires a lot of energy and the activity of an enzyme, flippase

60
Q

single-pass TM protein

A
  • type of integral membrane protein
  • has a single hydrophobic alpha helix that spans the phospholipid bilayer, leaving domains on the inheritor and exterior of the membrane
  • used to pass through membrane
  • ex: glycophorin A
61
Q

multi-pass TM protein

A
  • type of integral membrane protein
  • protein that spans the membrane more than once
  • ex: Bacteriorphodopsin
62
Q

beta-barrel

A
  • formed from 16 beta strands
  • used to span membranes
  • exterior is hydrophobic, interior is hydrophilic
  • ex: porins found in bacterial cells, chloroplast, and mitochondria
63
Q

channels

A
  • made from collection of alpha helices
  • exterior is hydrophobic, interior is hydrophilic
  • ex: aquaporin
64
Q

lipid-binding motifs

A

motifs containing amino acids that allow protein to interact with polar head groups of membranes

Biology 2B03 (5 decks)

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