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MCDB 428 > Exam 1 Flashcards > Flashcards

Exam 1 Flashcards

1
Q

What size are most tissue culture cells?

A

20-30 um

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2
Q

What sizes can light microscopy see?

A

from 25 nm to 10 mm

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3
Q

What kind of organism and structures can you see with light microscopy?

A
  • frog eggs
  • plant cell
  • animal cell
  • bacteria
  • viral ribosomes
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4
Q

What is the smallest organisim/structure that the conventional resolution of light microscopy can show?

A
  • bacteria
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5
Q

What is the smallest organisms/structure that super resolution of light microscopy?

A

viral ribosomes

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6
Q

What is transmission microscopy/ what are the mechanisms behind it?

A
  • white light that passes through a sample is reflected and deflected through other lens into the ocular eye piece
  • other light is absorbed or blocked by the sample, creating different planes/dimensions of the sample
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7
Q

What is epifluorescence microscopy? What are the mechanisms behind it?

A
  • a specimen is tagged with light of a specific wavelength
  • this light is absorbed and re-emitted fluorescence of a specific wavelength, which is collected in the eye piece
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8
Q

What is a major challenge in transmission microscopy?

A
  • maintaining or introducing contrast
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9
Q

What does the hematoxylin stain do?

A
  • basic dyes the nucleus
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10
Q

What does the eosin stain do?

A
  • acidic dyes that stains the cytosol
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11
Q

What are the major parts to the inside of a fluorescent microscope?

A

1) first barrier filter
2) beam-splitting mirror
3) second barrier filter

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12
Q

What does the first barrier filter do?

A
  • lets only blue light through with a wavelength between 450-490nm
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13
Q

What does the beam-splitting mirror do?

A
  • reflects light below 510 nm and transmits light above 510nm
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14
Q

What does the second barrier filter do?

A
  • cuts out unwanted fluorescent signals, passing specific green fluorescent emission between 520-560nm
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15
Q

How are antibodies used in fluorescence microscopy?

A
  • to recognize a protein
  • locate a particular structure
  • reveal subcellular compartments
  • cell dynamics
  • these are tagged by a fluorescent secondary antibody
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16
Q

What does DAPI stain for?

A
  • DNA/the nucleus
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17
Q

What are some different tags that are coupled to secondary antibodies?

A
  • fluorescent molecules for immunofluorescence
  • Gold beads for immune-electron microscopy
  • Enzymes for immunoblotting/ELISA (enzyme-linked immunosorbent assay)
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18
Q

What is GFP?

A
  • green fluorescent protein (238aa)
  • from bioluminescent jellyfish (Aequorea Victoria)
  • accepts blue light at a wavelength of 488nm
  • emits light at 509 nm as green
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19
Q

What is GFP used for?

A
  • used in live cell imaging once fused to organisms’ DNA
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20
Q

What can you use to study membrane protein degradation in live cells?

A

dual color staining/imaging

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21
Q

How does photo activity work in live cells?

A
  • Using light/energy you photoactivate a cell
  • If GFP or another fluorophore is tagged to your protein or molecule of interest, it will fluoresce in a certain spot
  • can visualize and follow its localization, movement, and degradation patterns
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22
Q

What is FRAP and FLIP?

A
  • FRAP = Fluorescence Recovery after Photobleach
  • FLIP = Fluorescence Loss in photobleaching
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23
Q

How does FRAP and FLIP work?

A
  • you fluorescently tag molecules of interest
  • use a strong laser to kill/remove a protein in a certain area with the fluorescent tag = photobleaching
  • you see overtime if the fluorescence/ molecule comes back
  • if it does = FRAP
  • if it doesn’t = FLIP
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24
Q

What is the physical limit of resolution for a light microscope?

A

~0.2um

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25
Q

Why is there a resolution limit on a light microscope?

A
  • visible light is 380-750nm (.38-.75um)
  • need to increase resolution to go lower
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26
Q

What is the resolution of a super resolution microscope?

A

~30-50nm

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27
Q

What is the resolution of an electron microscope?

A

0.1nm

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28
Q

What is the diffraction limit of a normal microscope?

A
  • 1/2 of the wavelength (on the x-y plane)
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29
Q

What is diffraction limit?

A
  • the maximum resolution
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30
Q

What are examples of super-resolution microscopies?

A
  • Structured Illumination microscopy (SIM)
  • Photo activated localization microscopy (PALM)
  • Stochastic Optical Reconstruction Microscopy (STORM)
  • Stimulated Emission Depletion (STED)
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31
Q

What does STORM do to fluorescence?

A
  • makes the lights less blurred and more pinpoint
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32
Q

How do PALM and STORM techniques work?

A
  • successive cycles of activation and bleaching allow well-separated single fluorescent molecules to be detected
  • add up the positions of each molecule to creat a map of the entire shape/structure
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33
Q

What is transmission electron microscopy (TEM)?

A
  • it illuminates samples with high energy electron waves instead of light waves
  • this leads to high-res. imaging because of the short wavelength of the electron beam, which is much much smaller than that of a photon
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34
Q

What are the advantages of EM?

A
  • high resolution (down to 0.1nm)
  • can see ribosomes and large protein complexes
  • can see membrane bilayer (~5nm wide)
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35
Q

What are the disadvantages of EM?

A
  • requires fixed/dead samples
  • use of electrons requires sample to be observed in a vacuum - if not, will get a lot of background noise
  • optimal imaging requires the use of heavy metal stains to provide contrast
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36
Q

How does immune-gold labeling work for EM?

A
  • use a secondary antibody that is conjugated with a gold particle
  • electron will be absorbed/attracted to gold particle
  • used to see specific structures and their location
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37
Q

What is Scanning electron microscopy (SEM)?

A
  • collects reflected electrons off the surface of samples
  • based on what angle they are reflected, creates images
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38
Q

What part does SEM and TEM visualize?

A
  • TEM can only see a cross section of a cell/structures
  • SEM can only see surfaces not inside structures
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39
Q

What is freeze-fracturing used for?

A

visualize the surfaces of internal structures

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40
Q

What is the process of freeze-fracturing?

A
  • specimen is immersed in liquid nitrogen at -196C
  • specimen is precisely pushed against a sharp blade
  • frozen tissue splits along lines of weakness, like in the middle of a membrane
  • fractured surfaces are etched with heavy metals so they can be seen
  • helps us to know what proteins are embedded in the membrane
  • enables organelles to be seen
  • gives better detail of specimen
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41
Q

What is the process of monoclonal antibody production?

A
  • immunize a model organism (mouse) with a specific antigen
  • this makes B-lymphocytes that produces a variety of antibodies against the antigen
  • combine B lymphocytes and an immortalized tumor cell line.
  • add with a fusing agent, then centrifuge
  • causes the formation of heterokaryons, which are cultured in selective media
  • over time in culture, these cells become hybrids and are then clones to produce monoclonal antibodies
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42
Q

What is velocity sedimentation?

A
  • separates particles based on their relative size M(r)
  • an example of this is cell fractionation
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43
Q

What is M(r)?

A
  • molecular radius
  • a combination of both size and density
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44
Q

What is the process of velocity sedimentation?

A
  • sucrose or another high density liquid is used to increase the amount of time it takes for particles to sediment at the bottom
  • helps your sample to separate at a greater resolution for particle sizes
  • fast-sedimenting (bigger molecules) are lower than smaller, slow-sedimenting molecules
  • fractionate by separating drops or portions of your sample
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45
Q

What is differential centrifugation?

A
  • uses increasingly faster speeds to separate different particle groups
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46
Q

What structures do you receive after low-speed centrifugation (1000 x g, 10min)?

A

larger structures:
- whole cells
- nuclei
- cytoskeletons

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47
Q

What structures do you get after medium-speed centrifugation (20,000 x g, 20min)?

A

smaller organelles:
- mitochondria
- lysosomes
- peroxisomes

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48
Q

What structures do you get after high-speed centrifugation (80,000 x g, 1hr)?

A
  • microsomes
  • small vesicles
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49
Q

What structures do you get after very high-speed centrifugation (150,000 x g, 3hr)?

A
  • ribosomes
  • viruses
  • large macromolecules
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50
Q

What is an equilibrium gradient?

A

separates structures of different densities, not size

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51
Q

How does the protein composition of different organelles affect the results of doing an equilibrium gradient?

A
  • the presence of distinct proteins in each organelle increases their densities differently
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52
Q

How does an equilibrium gradient work?

A
  • your create a solution with a density gradient, like a sucrose gradient
  • load sample on top and centrifuge at a high speed
  • molecules with lower densities (lipids) will on top
  • molecules with higher densities (proteins) will be towards the bottom
  • fractionate out different layers by various drops
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53
Q

What does SDS-page gel do?

A

separates proteins based on their sizes
- linearizes the protein

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54
Q

How do antibodies function in a western blot?

A
  • primary antibody attaches to a specific protein of interest
  • secondary attaches to the primary and is conjugated with either a fluorophore or an enzyme to catalyze a light reaction
  • these conjugations help with visualization
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55
Q

What is the purpose of a pulse-chase experiment?

A
  • to study the life span, trafficking, modification and death of a protein
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56
Q

What is the pulse phase in a pulse-chase experiment?

A
  • chemically tag newly synthesized proteins
  • tag is to metabolically label with radioactive amino acids
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57
Q

What is the chase phase in a pulse-chase experiment?

A
  • after initial labeling of a specific protein during the pulse phase, block further labeling to follow the protein
  • do a time course analysis to study:
    where its going
    modifications that are happening
    and protein turnover
  • this can be done using a western blot for the protein of interest in different organelles
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58
Q

What is forward genetics?

A
  • isolate mutants that have a phenotype, then identify the genes behind it
  • this can be done by a genetic analysis or screening of a DNA library, then expressing a potential gene in culture
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59
Q

What is reverse genetics?

A
  • finding a gene of interest and then mutating the gene and finding a phenotype
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60
Q

What is the myostatin gene?

A
  • a gene that regulates muscle development and mass
  • when mutated, you get an abundance of muscle mass
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61
Q

How can conditional mutations be used to study essential genes?

A
  • an example of this is studying temperature response genes in yeast and bacteria.
    -mutant cells that are temperature sensitive are incubated at 36C and 23C
  • cells at 23C proliferate because proteins are able to fold at lower temps better
  • cells at 36C did not proliferate, to hot for their proteins to fold
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62
Q

What does a complementation analysis do?

A
  • to see if 2 mutations that produce the same phenotype are on the same gene
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63
Q

How does a complementation analysis work?

A
  • haploid games for each mutant are mated to form diploids
  • plate these organisms
  • if one set of diploid cells don’t grow, then they are on the mutations are within the same gene
  • if the organisms do grow, that means the the mutations are on different genes and are complementary because their genes were able to compensate for another and cause normal function
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64
Q

What is the point of RNAi/siRNA?

A
  • to knockdown or knockout genes:
  • cleaves target RNA
  • translation repression and destruction of target RNA
  • formation of heterochromatin on DNA from which target RNA is being transcribed.
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65
Q

How does an RNAi experiment work?

A
  • take dsRNA that is complementary to target RNA seq (found in many viral genomes)
  • it gets processed into a single 23bp stranded in a argonaut or piwi protein
  • matches with complementary RNA and effects protein production
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66
Q

What does CRISPR/Cas9 do?

A
  • adapted from bacteria to help edit genomes
  • causes dsDNA breaks
  • can repress or activate gene expression
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67
Q

How does the CRISPR/Cas9 system work?

A
  • Cas9 protein comes from bacteria and recognizes a specific RNA sequence (guide RNA)
  • contains a guide RNA to match a specific sequence/location in the genome
  • used to find gene of interest/modify it
  • once the guide RNA/Cas9 structure recognizes the PAM sequence, it can cleave it as an endonuclease
  • if Cas9 is fused with a transcription activator, can activate gene expression near the PAM sequence, or do the opposite if it is fused with a repressor domain (can’t cut the DNA like this)
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68
Q

What are the characteristics of proteins?

A

1) proteins carry out most cell functions
2) polymer of up to 20 different subunits (AAs)
- identical chemistry of joining (peptide bond)
- no branching
- only order and number of AA vary
3) a lot of diversity in structure and function

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69
Q

What is the secondary structure of proteins

A
  • regular repeating arrangement of backbone + hydrogen bonds
  • 3D segments called domains
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70
Q

What is protein tertiary structure?

A
  • packing of helices/sheets in 3D of an entire
  • called conformation/shape
  • contains both secondary structures and unstructured domains
  • primarily stabilized by hydrophobic interactions
    ~ 60% of protein consists of ordered secondary structure and the remainder is disordered
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71
Q

What is protein quaternary structure?

A
  • fitting multiple polypeptides together
  • association of multiple polypeptides to form complexes
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72
Q

What is macromolecular assembly?

A
  • regulated assembly of proteins, large complex or protein machine
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73
Q

Is cysteine polar or non polar?

A
  • nonpolar
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74
Q

What are characteristics of hydrophobic amino acids?

A
  • tend to stay on the inside of folded proteins to keep away from the hydrophilic environment
  • form transmembrane domains to span lipid bilayers
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75
Q

What is special about the hydrophobic tyrosine amino acid?

A
  • contains a hydroxyl group on its benzyl ring
  • can be phosphorylated during signaling
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76
Q

What are the characteristics of Alanine?

A
  • Ala
  • A
  • non polar/hydrophobic
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77
Q

What are the characteristics of Glycine?

A
  • Gly
  • G
  • non polar/hydrophobic
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78
Q

What are the characteristics of Valine?

A
  • Val
  • V
    non polar/hydrophobic
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79
Q

What are the characteristics of Leucine?

A
  • Leu
  • L
  • non polar/hydrophobic
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80
Q

What are the characteristics of Isoleucine?

A
  • Ile
  • I
    non polar/hydrophobic
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81
Q

What are the characteristics of Proline

A
  • Pro
  • P
    nonpolar/hydrophobic
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82
Q

What are the characteristics of Phenylalanine?

A
  • Phe
  • F
    non polar/hydrophobic
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83
Q

What are the characteristics of Methionine?

A
  • Met
  • M
    nonpolar/hydrophobic
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84
Q

What are the characteristics of Tryptophan?

A
  • Trp
  • W
    nonpolar/hydrophobic
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85
Q

What are the characteristics of Cysteine?

A
  • Cys
  • C
    nonpolar/hydrophobic?
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86
Q

What are the characteristics of Aspartic acid?

A
  • Asp
  • D
    negatively charged
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87
Q

What are the characteristics of glutamic acid?

A
  • Glu
  • E
    negatively charged
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88
Q

What are the characteristics of Arginine?

A
  • Arg
  • R
    Positively charged
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89
Q

What are the characteristics of Lysine?

A
  • Lys
  • K
    positively charged
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90
Q

What are the characteristics of Histidine?

A
  • His
  • H
    positively charged
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91
Q

What are the characteristics of Asparagine?

A
  • Asn
  • N
    polar
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92
Q

What are the characteristics of Glutamine?

A
  • Gln
  • Q
    polar
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93
Q

What are the characteristics of Serine?

A
  • Ser
  • S
    polar
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94
Q

What are the characteristics of Threonine?

A
  • Thr
  • T
    polar
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95
Q

What are the characteristics of tyrosine?

A
  • Tyr
  • Y
    polar
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96
Q

What are the characteristics of hydrophilic Amino acids?

A
  • typically exposed to water
  • Ads with similarly charged side groups tend to repel one another
  • salt bridges can form between positively and negatively charged amino acids
  • lysine can get a ubiquitin attachment
  • Asparagine can get glycosylated
  • polar groups can get phosphorylated
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97
Q

What is special about Cysteine?

A
  • can form intra or inter-molecular disulfide bonds
  • site of post-translational fatty acylation (prenylation. palmitoylation, farnesylation) of membrane associated proteins
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98
Q

What is special about glycine?

A
  • has a hydrogen as its side group
  • this makes it packs well and often to save as a flexible helix breaking residue
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99
Q

What is special about proline?

A
  • side chain bends backwards to form a ring
  • limits the ability of the polypeptide chain to rotate in the vicinity of this residue
  • another helix breaking residue
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100
Q

What is special about aromatic AAs?

A
  • can be used to calculate protein concentration
  • Trp rings absorb the most UV, then tyrosine, then phe
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101
Q

What amino acids can be phosphorylated?

A
  • ser, thr and tyr
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102
Q

What AAs can be glycosylated?

A
  • Ser and Thriller can be O-linked glycosylated in the Golgi lumen and in the cytosol
  • Asn can be N-linked glycosylated in the ER
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103
Q

What is the MW of an amino acid?

A
  • 110 Daltons / 110 grams per mole
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104
Q

What are the characteristics of polypeptides?

A
  • peptide bonds are planar due to the resonance of C=O and the C-N bonds
  • because of this, rotation can only occur around side groups
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105
Q

What are the non covalent bonds that help with protein folding?

A
  • electrostatic attractions
  • hydrogen bonds
  • van der Waals attractions
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106
Q

Why are some 3D structures stable?

A
  • hydrogen bonding of backbones amide and the carbonyl groups
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107
Q

T/F: secondary structures does not generally depend on the types of amino acids to comprise structure

A

true

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108
Q

What are the 3 main secondary structures for proteins?

A
  • alpha helices
  • beta sheets
  • beta turns
  • proteins fold into a conformation of the lowest energy
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109
Q

How many degrees does each amino acid turn?

110
Q

How many amino acids are in every turn of a helix?

111
Q

What is the distance between amino acids 1 full turn and on the same face of a helix?

112
Q

Where does hydrogen bonding occur to stabilize and alpha helix?

A
  • between the N-H and C=O of peptide bonds 4 residues apart
113
Q

How many AAs are typically in 1 a helix?

A

7 = two full turns

114
Q

How are a helices arranged?

A
  • side. chains are pointed out.
  • can be amphipathic by one side being hydrophobic, and another side is hydrophilic
115
Q

What interactions do a helices take part in normally?

A
  • protein-protein
  • are usually what consists of transmembrane-spanning domains (18-22AAs long when no proline present)
116
Q

What happens if there is a proline or glycine present in an a helix?

A
  • it would break into 2 helices
117
Q

How many AAs in a strand of a beta sheet?

A
  • 5-8 residues
118
Q

Where does hydrogen bonding occur in beta sheets?

A
  • occurs between strands, not adjacent AAs
  • side chains stick out above and below strands
119
Q

How are the strands arranged?

A

can be either parallel or anti-parallel

120
Q

What is circular dichroism?

A
  • the way left circular and right circular polarized lights are absorbed differently by different secondary structures
121
Q

How is circular dichroism in protein secondary structure analysis?

A
  • measuring absorbances of the different light polarities can be used to estimate the content of secondary structures
122
Q

What are the 3 main classes of tertiary structure?

A
  • globular
  • fibrous
  • transmembrane
123
Q

What is a structural motif?

A
  • a small structure signature ( a couple AAs long)
  • can be recognized by various protein
  • is small, folding depends on surrounding sequence
    Ex: NxS motif could be an N-glycosylation site on secretory proteins
124
Q

What is a structural domain?

A

-Portion of a protein (10-100 AAs)
- has a tertiary structure of its own
- large proteins normally have more than one domains that are connected by flexible linkers

125
Q

What is a coiled coil?

A
  • made up of heptad (7) repeats
  • this is because its usually 2 full turns
  • the same amino acid every 7 AAs on the same face
  • hydrophobic residues on the inside facing each other
  • to sets of helices around each other
126
Q

What is an EF-hand domain?

A
  • helix-loop-helix domain
  • used for DNA binding
  • is the domain used to bind to calcium, coordinated by the negatively charged Asp and Glu residues
127
Q

What is a Zn-finger domain?

A
  • DNA binding domain
  • zinc binds, coordinated by two pairs of His and Cys residues
128
Q

What is a structural domain?

A
  • combination of secondary structures used for a specific kind of function
    -distinct region of a tertiary structure
129
Q

What has been developed and used over recent years to predict protein folding with >90% accuracy?

A
  • AI (alpha fold)
130
Q

What are the main roles of membranes?

A
  • separate all cellular processes from the outside environment
  • maintain compartmentalization of different organelles
131
Q

What constitutes a membrane?

A

50% lipids + 50% proteins
- molar ratio 50:1

132
Q

What are the 3 main types of lipids?

A
  • phosphoglycerides (PC,PE, PS)
  • sphingolipids
  • sterols
  • phosphinositides are less abundant but prevalent to signaling
    ** also glycolipids
133
Q

What are the characteristics of phosphoglycerides?

A
  • Has a glycerol backbone:
    a) two carbons have oxygens each attached to a fatty acid tail, carbon backbone is not directly attached
    b) third carbon is bonded to a phosphate group + another characteristic group depending on the glyceride
  • fatty acids can either be saturated our unsaturated
134
Q

What are the characteristics of PE?

A

phosphatidylethanolamine
-phosphoglyceride
- has an ethanol amine group
- net neutral charge
(negative charge of phosphate group + positive charge on the amine group cancel out)

135
Q

What are the characteristics of PS?

A

phosphatidylserine
- phosphoglyceride
- has a serine group attached to the phosphate
- net negative charge ( negative charges of the phosphate and carbonates + the positive charge of the amine group)

136
Q

What are the characteristics of PC?

A

Phosphatidylcholine
- phosphoglyceride
- has a choline group attached to the phosphate
- net neutral charge (negative charge of phosphate group + positive charge on the amine group cancel out)

137
Q

What are the characteristics of the sphingosine backbone?

A
  • first carbon is directly attached to a Fatty acid chain, with a hydroxyl group separately attached
  • second carbon contains an amine group which can be used to attached other groups
  • third carbon has a hydroxyl group that can bind to a phosphate + characteristic group depending on the type of sphingolipid
138
Q

What are the characteristics of sphingomyelin?

A
  • sphingolipid
  • amine group is attached to a carbonyl group with a fatty acid tail
  • has a choline group attached to the phosphate
  • net neutral charge
139
Q

What 4 types of lipids compose a majority of the mammalian membrane?

A
  • PE
  • PS
  • PC
  • SM
140
Q

What is the common structure of most sterol?

A
  • a 4 ring hydrocarbon group
141
Q

What are the characteristics of cholesterol?

A
  • there is a hydroxyl group attached to an end of the hydrocarbon ring group
    -has a short nonpolar/hydrophobic hydrocarbon tail
  • these make it small and amphipathic
  • can’t form a bilayer on its own, but is small enough to intercalate between other lipids
  • synthesized by the ER and primarily localized in the PM
  • can also come from food, which the lysosome can recycle and separate
142
Q

What are the common characteristics of glycolipids?

A
  • located on the outer leaflet of membranes
  • sugar groups are added to lipids in the Golgi lumen
  • has sugar head groups instead of phosphates
  • some sugars can be charged
  • typically uses the sphingosine backbone
  • glycolipids in bacteria have a glycerol backbone
143
Q

What is galactocerebroside?

A
  • glycolipid
  • major in mammalian lipid bilayers
  • sphingosine backbone
  • has a galactose group attached
144
Q

What is ganglioside?

A
  • glycolipid
  • sphingosine backbone
  • has 5 sugar groups attached at the third carbon
  • negatively charged NANA group
145
Q

What are common characteristics of phosphoinositides?

A
  • low abundance lipids
  • plays a big role in cell signaling
  • has a glycerol backbone
  • phosphate is attached to an inositol ring
    a. has 5 hydroxyl groups in the ring
    b. 3,4 and 5C hydroxyl groups can be differently phosphorylated depending on the purpose/intended location of the PI
    -different combinations of phosphate groups on 3,4,5 OH = 7 total phosphoinositides
146
Q

T/F: most lipids are amphipathic

147
Q

What structures do lipids normally form into?

A
  • micelle, especially when the shape of the lipid is like a cone
  • and lipid bilayer (cylinder shape lipid, ex: phospholipids)
  • depends on the environment and what is energetically more favorable in the environment
148
Q

How do lipids move most commonly?

A
  • laterally diffuse the most
  • fluidity depends on its composition and temperature
  • flipping across layers rarely occurs
149
Q

What happens with the membrane at lower temperatures?

A
  • bilayers become more Gel-like and stiff
  • increases amount of unsaturated FAs to allow for more movement/less rigidity to prevent freezing
  • saturated FAs move slower because they are more stiff
150
Q

T/F: transmembrane proteins can’t laterally diffuse within a membrane

A

false: they can

151
Q

What enzyme allows for lipids to flip between bilayers of membranes?

152
Q

True or False; the lipid bilayers are asymmetrically composed of different lipid types.

153
Q

What types of lipids are prevalent on the extracellular layer of the plasma membrane?

A
  • Phosphatidylcholine
  • sphingomyelin
  • glycolipids
  • typically the heavier layer is oil the outside membrane
154
Q

What types of lipids are prevalent on the cytosolic layer of the plasma membrane?

A
  • Phosphatidylethanolamine
  • Phosphatidylserine
  • phosphoinositides
  • phosphoinositide phosphates
155
Q

What side of the plasma membrane would the lumen side of a transmembrane protein face?

A
  • extracellular face
156
Q

T/F: both lipids and proteins can be glycosylated

157
Q

What forms a heavy layer on the outer membrane to form a protective barrier around the cell?

A
  • glycosylation of membrane proteins, lipids, and carbohydrates
158
Q

Where do lipids come from?

A
  • the ER
  • packaged and transported in and from the golgi
159
Q

Where are the enzymes that glycosylate proteins and lipids?

160
Q

Why would it be important for PIPs (Phosphoinositides phosphates) to be on the cytosolic side of the plasma membrane?

A
  • to be easily accessible for their major role as signaling molecules
161
Q

What are the types/ways that different proteins associate with membranes?

A
  • integral, lipid-anchored
  • peripheral membrane proteins (other forces attract it to the membrane)
  • single or multi-pass transmembrane alpha helices
  • beta barrel structures
  • amphipathic alpha helices (lay horizontal in the lipid membrane)
  • can be anchored by lipid modifications (prenylations or a GPI anchor)
162
Q

How can peripheral proteins be extracted?

A
  • with a solution that has a pH >10 or high pH salt
163
Q

How can GPI-anchored proteins be extracted?

A
  • released by using phospholipase C (PLC)
164
Q

how can integral membrane proteins be extracted from the plasma membrane?

A
  • can only be released with detergents
165
Q

What are the characteristics of a hydrophobic alpha helices region of a protein on a hydropathy plot?

A
  • has a peak in the positive area
  • is >18 amino acids long
166
Q

What determines the different types of a lipid anchor?

A
  • how many carbons are in the fatty acid chain
  • where on a protein is the anchor attached
167
Q

What are the characteristics of myristoylation/myristoyl anchors?

A
  • 14C fatty acid chain
  • attached to the first glycine on the N-terminus end of a protein
168
Q

If methionine is the first amino acid to signal the start of protein translation, how are other amino acids like glycine, found at the end terminus end?

A
  • prior amino acids are cleaved off
169
Q

What are the characteristics of palmitoylation anchors?

A
  • 16C fatty acid chain
  • attaches to a cysteine on the protein
  • thioester linkage
170
Q

What are the characteristics of farnesylation anchors?

A
  • 15C fatty acid chains
  • forms a thioester bond between the prenyl group and a cysteine on the protein
171
Q

What is the common face that myristoyl, palmitoyl and farnesyl anchors are on?

A

the cytosolic face

172
Q

What are the characteristics of a GPI anchor?

A
  • glycophosphatidylinositol
  • attaches to the C-terminus end of the protein
  • similar to a glycosphingolipid, but the head group is an inositol instead
  • has a glycerol backbone
173
Q

What direction do GPI anchors face?

A
  • because these are constructed and attached in the ER lumen, they always face outside of the cell
174
Q

How can GPI-anchored proteins be released from the membrane?

A
  • using an inositol-specific phospholipase C enzyme
175
Q

What are all the parts of a GPI anchor?

A
  • glycerol backbone
  • phosphate group
  • inositol
  • glucosamine (1)
  • mannose (3)
  • ethanolamine
176
Q

What is the process of reconstituting membrane proteins into liposomes?

A

1) add detergent to break apart the membrane
2) purify membrane proteins that have solubilize on their own from other lipid clumps
3) add phospholipids mixed with detergent
4) remove detergent, which allows the phospholipids and membrane proteins to form liposomes

177
Q

What is the order of most permeable to least permeable molecules across a membrane?

A
  • hydrophobic molecules (gases with no charge)
  • small uncharged polar molecules
  • large uncharged polar molecules
  • ions
178
Q

Is the Na+ concentration higher inside or outside the cell?

A

outside > inside

179
Q

is the K+ concentration higher on the inside or outside of the cell?

A

inside > outside

180
Q

Is the Mg2+ concentration higher on the inside or outside?

A

outside > inside

181
Q

Is the Ca2+ concentration higher on the inside or outside of a cell?

A

outside > inside

182
Q

Is the proton concentration higher on the inside or outside of the cell?

A

inside > outside

183
Q

Is the Cl- concentration higher on the inside or outside of the cell?

A

outside > inside

184
Q

What is the largest family of membrane transporters?

A

ABC-transporters

185
Q

What role does membrane transport play in Cystic fibrosis and CTFR?

A
  • doesn’t allow for the adequate transport/secretion of pancreatic enzymes
186
Q

What role does membrane transport play in bile salt transporter disorders?

A
  • ABC transporters in the liver inadequately transport or allow the secretion of bile
187
Q

What role does membrane transport play in retinal degeneration?

A
  • ABCA4 transporter doesn’t allow the passage retinol into the cells
188
Q

What role does membrane transport play in mitochondrial iron homeostasis?

A
  • ABCB7 transporter is mutated, causing anemia and ataxia
189
Q

What role does membrane transport play in multi drug resistance?

A
  • ABC family transporters export drugs out of the cell to prevent harmful effects
190
Q

What are the causes/characteristics of Wilson’s disease?

A
  • ATP7B transports cytoplasmic copper into the Golgi lumen in cells inside the liver
  • high copper concentration in the cytoplasm is known to do extensive damage
  • can’t have chocolate :(
191
Q

What are the causes/characteristics of Niemann-Pick Disease type C?

A
  • cholesterol is pulled into the cell and goes into the lysosome
  • cholesterol is broken down into free sterol/cholesterol + a Fatty Acid
  • MPC-1 transporting protein on the lysosomal membrane normally transports free cholesterol to allow it to be used
  • in NPD, MPC-1 is broke and cholesterol stays in the lysosome and causes it to build up and make the lysosome nonfunctional
192
Q

What are the 2 main types of membrane transport?

A
  • simple, passive diffusion
  • mediated transport
193
Q

What types of molecules undergo simple diffusion?

A
  • organic molecules
  • small uncharged molecules
194
Q

What are the 2 subtypes of Mediated transport?

A
  • passive transport
  • active transport
195
Q

What is passive transport?

A
  • facilitated diffusion and channels
  • solutes go from high to low concentration
196
Q

What is active transport?

A
  • ATP powered/energy powered pumps and transporters
  • from low to high solute concentration
197
Q

Is membrane transport or diffusion faster?

A
  • membrane transport
198
Q

What limits membrane transport?

A
  • the amount of proteins/enzymes to mediate transport
  • can become saturated and display a Vmax
199
Q

What can change the speed of diffusion?

A
  • substrate concentration
200
Q

what are the general characteristics of membrane transporters?

A
  • undergoes a conformational change during transportation
  • relatively slow
201
Q

What are the general characteristics of channel proteins?

A
  • a pore
  • always downhill for concentration gradients
  • super fast
202
Q

What about a transporter mediates the passive movement of a solute?

A

conformational change of the transporter
- transition occurs randomly and are reversible

203
Q

What is a requirement of all forms of facilitated transport?

A
  • need a pre-formed gradient
204
Q

What are the 3 kinds of facilitated transport?

A
  • uniport
  • symport
  • antiport
205
Q

What type of transporter is GLUT1?

A
  • a glucose uniporter
  • ## goes from high concentration outside the cell to low concentration inside the cell
206
Q

What is Km in relation to GLUT1?

A
  • the concentration of glucose at which half of GLUT1 in the membrane are bound to glucose
207
Q

What changes the conformation of GLUT1 to allow glucose to enter the cell?

A
  • the binding of glucose in the outward-facing conformation
  • can be reversed when glucose is released into the cell
208
Q

What is the Km for glucose?

209
Q

Why are cytosolic levels of glucose so low and keep bringing more glucose in?

A
  • glucose is rapidly converted to G6P in the cell, bringing down the glucose concentration
210
Q

What are the 3 main types of active transport membrane transporters?

A
  • coupled transporters: anti port and symporter
  • ATP-driven pump: ATPase
  • light-driven pump
211
Q

When can the conformation change occur in a symporter?

A
  • only when all binding sites are fully occupied or completely empty
212
Q

In which cases would it be difficult to bring glucose into the cell?

A
  • cytosolic glucose concentration is high
    (like in small intestinal epithelium)
213
Q

What type of transporter is used in the case of there being a high glucose concentration in the cytosol but you still want to bring glucose in?

A
  • Na+- linked glucose symporter
  • both Na+ and glucose is coming into the cell
  • linked to energetically favorable transport of another ion from the formed electrochemical gradient
214
Q

What are the characteristics of the Na+-linked glucose transporter?

A
  • glucose + 2Na+ ions are binding to the symporter (cooperative)
  • causing the conformational change and release of molecules into the cytoplasm
215
Q

What makes the reverse reaction of symport of 2Na+ and glucose out of the cell?

A
  • this reaction is still reversible
  • the Low cytosolic Na+ concentration makes it less likely to occur tho
216
Q

What are the 3 main families of ATP- pumps?

A
  • p-type pump
  • ABC transporter
  • V-type and F-type pumps/synthases
217
Q

What are the characteristics of the P-type ATP pump?

A
  • phosphorylates themselves during transport, like ATP7B
  • phosphorylation and dephosphorylation powers conformational changes
218
Q

What are examples of P-type pumps?

A
  • a proton pump on the plasma membrane of plants fungi and bacteria
  • plasma membrane of higher eukaryotes (Na+/K+)
  • apical plasma membrane of the mammalian stomach (H+/K+)
  • Plasma membrane of all eukaryotic cells (Ca+ pump)
  • Sarcoplasmic reticulum membrane in muscle cells (Ca2+ pump)
219
Q

What are the general characteristics of the Na+/K+ P-type ATPase?

A
  • 3Na+ out and 2K+ in against both of their concentration gradients
  • blocked by ouabain
  • consumes 1/3 of energy in mammalian cells to maintain a electrochemical gradient in cells
220
Q

What are the general characteristics of the Na+/K+ ATPase transport cycle?

A
  • transient catalytic phosphorylation
  • 2 conformational changes: E1 and E2
  • Na+ dependent phosphorylation
  • K+ dependent dephospho rylation
221
Q

What is the transport cycle process of the Na+/K+ ATPase?

A

1) 3Na+ enters the transporter that is in the closed position on the cytosolic side
2) ATP transfers a phosphorylation to the pump, causing the first conformation change
3) the open extracellular face of the transporter allows for the Na+ molecules to be released
4) binding of 2K+ triggers release of phosphate group, relaxing the pump and turning it back to the initial conformation (change 2)
5) K+ is released into the cell

222
Q

What specific cell type is the sarcoplasmic reticulum found in?

A

a special type of ER in muscle cells

223
Q

What is the general characteristics of the SR Ca2+/ATPase pumping cycle?

A
  • calcium dependent phosphorylation
  • proton dependent dephosphorylation
  • majority of the membrane proteins in the SR
224
Q

What is the pumping cycle process of the SR Ca2+ ATPase?

A
  • ATP is bound to the ATPase on the cytosolic side
  • pump is open to the cytosolic face, allowing 2 calcium ions to enter
  • this causes the conformation to change, causing the cytosolic side to close and ATP to phosphorylate the pump on an Asp residue, turning into ADP
  • ADP leaves the complex, replaced by another ATP molecule
  • upon ATP binding, causes a conformational change to open the pump on the SR lumen side, release the calcium
  • 2 protons enter the pump and causes the lumen side to close
  • this conformational change causes the release of the phosphate group
  • this opens the pump to the cytosolic face and allows the protons to leak into the cytoplasm
225
Q

What are some common examples or cases of V-type ATP pumps?

A
  • used in vacuolar membranes in plants, yeasts and other fungi
  • endosomal and lysosomal membranes in animal cells to acidy them
  • plasma membrane of osteoclasts and some kidney tubule cells
226
Q

What is the main role of V-type pumps?

A
  • pumps protons out of a cell across a membrane using ATP as energy
227
Q

What are common examples of F-type ATP pumps?

A
  • bacterial plasma membrane
  • inner mitochondrial membrane
  • thylakoid membrane of chloroplasts
228
Q

What is the main role of F-type ATP pumps?

A

synthesizes ATP by bringing in protons down its concentration gradient across a membrane

229
Q

What are some common examples of ABC transports as ATP pumps?

A
  • bacterial plasma membranes to transport AAs, sugars and peptides
  • Mammalian plasma membranes to transport phospholipids, drugs and cholesterol
230
Q

How does ATP binding work in the CFTR Cl- channel?

A
  • ATP binding and hydrolysis drives the opening and closing of the Cl- channel instead of transporting ions across a membrane
231
Q

What are the 4 types of gated ion channels?

A
  • voltage-gated (K+ channels)
  • extracellular ligand-gated (acetylcholine receptors)
  • intracellular ligand-gated (IP3-gated Ca2+ channel)
  • mechanically gated (membrane tension linked to cytoskeleton)
232
Q

What are the major differences between membrane transporters and channels?

A
  • conformation change during the passage of solutes (transporters only)
  • channels are much faster than transporters
233
Q

What do small conformation changes allow channels to/what do they change?

A
  • when channels are closed, 4 inner alphas helices are close together, making the selectivity filter/pore to small
  • when the conformation changes to open the channel, 4 inner helices move apart to open the pore more
234
Q

What are the characteristics of the K+ channel structure?

A
  • water-lined pore through the membrane
  • selectivity filter that allows only specific ions through (K+)
  • H2O has to be removed from the K+ to pass so the ion can pass through the selectivity filter
  • the dehydrated K+ ions interact with the carbonyl oxygens of amino acids on the selectivity loop when it passes through the filter
235
Q

How does the pore helix help to draw in K+ ions through the filter or into the channel?

A
  • the negative dipole end is point at the inner pore where K+ will pass through. Opposite charge draws K+ through
236
Q

What kind of Amino acids line the membrane faces of the inner and outer helices of the K+ channel?

A

negatively charged AAs to draw in the positively charged K+ ions

237
Q

What is the net charge on the cytosolic side of the cell?

238
Q

What is the net charge on the extracellular side of the cell?

239
Q

How do all excitable cells generate action potentials

A

voltage-gated cation channels

240
Q

What are the 4 parts of a voltage-gated Na+ channel?

A

1) voltage sensors
2) central channel
3) selectivity filter
4) inactivation gate

241
Q

What is the voltage sensor on a voltage-gate cation channel/what does it do?

A
  • has (+) arginines as one of the transmembrane helices
  • attracted to the negatively charged extracellular membrane during depolarization
  • depolarization causes conformational change of the channel, opening the pore
242
Q

What is the point of the refractory state?

A

-inactivation gate plugs the pore
- this stops backwards and reactivation of channels
- stays there until after the membrane is repolarized

243
Q

When does the channel become inactivated by the inactivation gate?

A
  • when the equilibrium potential is reached
244
Q

What is propagation of action potentials?

A
  • signals maintain strength by working in one direction
  • channels are opened one-by-one as nearby action potentials are depolarized
245
Q

What is the process of Na+/K+ ATPase action potential and depolarization?

A
  • ATPase sets up the Na+ and K+ gradients (NA out of the cell and K into the cell)
  • Na+ channels open upon depolarization
  • inactivation and closing in the lagging direction
  • K+ leak channels depolarize the membrane (goes out of the cell
246
Q

What is the use of a Patch-Clamp?

A
  • used to record individual ion channels
  • you use a glass micropipette with gentle suction
  • pulls about individual ion channels off of the membrane
247
Q

What allows for an increased speed of action potentials in axons?

A
  • layers of myelin sheaths help to keep the ions localized
  • this helps to maintain membrane potentials = allows for action potential to be used faster and maintained
  • Schwann cells (glial cells) also cover the axon to do this
248
Q

What is the process of transmitting a signal across a neuromuscular junction?

A

1) action potential depolarizes and opens voltage-gated calcium channel, bringing calcium into the presynaptic nerve
2) Calcium uptake causes the release of acetylcholine into the synapse space
3) acetylcholine binds to Na+ channels, causing and influx of Na+, depolarizing the post-synaptic membrane
4) this depolarization causes the opening of Na+ voltage-gated channels, increasing the influx of Na+
5) This causes the activation and depolarization of voltage-gated calcium channels in the T tubule
6) the conformational change of this channel opening causes tension in Calcium channels on the SR = opening and release of calcium into the cytosol
7) cytosolic calcium binds to troponin and causes the muscles to contract

249
Q

What are the two signaling pathways that translated proteins can take?

A
  • co-translational
  • post-translational
250
Q

What is the general process of the secretory pathway in the cell?

A

1) translation begins, producing a signal sequence
2) signal sequence targets the ribosome-protein complex for the ER
3) ribosome co-translates the peptide against the ER membrane, pushing the peptide into the ER lumen or on the membrane
4) secreted from the ER into the Golgi
5) packaged and modified in the golgi, then is secreted to either the plasma membrane or the lysosome

251
Q

Where do post-translationally targeted proteins tend to go?

A
  • nucleus
  • mitochondria
  • plastids
  • peroxisomes
252
Q

Where to co-translationally targets proteins tend to go?

A
  • plasma membrane
  • tonoplasts
  • secreted proteins
  • cell wall
  • vacuole
253
Q

What are the 3 ways that proteins move between compartments in the cell?

A
  • gated transport
  • protein translocation across the membrane
  • vesicular transport
254
Q

What is gated protein transport?

A
  • movement of proteins and RNA between the cytosol and nucleus
  • mainly folded proteins
255
Q

What is protein translocation across the membrane?

A
  • proteins moves into the ER and mitochondria, chloroplast
  • needs to unfold and fold proteins
256
Q

What is vesicular protein transport?

A
  • proteins do not cross a membrane
  • mainly folded proteins
257
Q

What parts of the cell undergo gated transport?

A
  • between the cytosol and nucleus
258
Q

What parts of the cell undergo protein translocation across the membrane?

A

Cytosol to:
- plastids
- mitochondria
- ER
- peroxisomes

259
Q

What parts of the cell undergoes vesicular transport?

A
  • peroxisomes
  • ER
  • Golgi
  • Late endosome
  • lysosome
  • early endosome
  • cell exterior
260
Q

What is a main characteristic of nuclear pores?

A
  • spans both membranes
  • nuclear envelope is continuous with ER membrane
261
Q

What is the role of cytosolic fibrils?

A
  • grabs incoming cargo
262
Q

What is the ring of the nuclear pore like?

A
  • octagonal
  • 8-fold rotational symmetry
263
Q

What are the characteristics of gated diffusion of nuclear pore complexes?

A
  • small molecules enter through via free diffusion
  • macromolecules >60kDa have to be unfolded unless there is energy consumption to send it through
264
Q

What are the characteristics of nuclear transport?

A
  • post-translational, bidirectional
  • fully folded proteins can be transported
  • transport occurs across a large, expandable aqueous pores
  • reversible, but regulated
  • 9nm pore
  • can expand for ribosomal subunits, but not mature ribosomes
265
Q

What is the signal sequence like when the protein is being imported into the nucleus?

A
  • positively charged amino acids (Arg, Lys)
266
Q

What are the signal sequences like on proteins that are being exported from the nucleus?

A
  • leucine rich, amphipathic
  • Met, leu, Phe
267
Q

What is the nuclear localization sequence (NLS)?

A
  • nuclear import sequence
  • one or more short, internal sequences of K or R residues (positively charged)
  • can be found in any part inside the protein
268
Q

What are characteristics of NLS?

A
  • the precise sequence varies for different nuclear proteins
  • can be anywhere in the proteins
  • positive charge
  • both necessary and sufficient for nuclear import
269
Q

What are the characteristics/ process of the NLS-gold experiment?

A
  • gold particles are coated with NLS-containing peptides and injected into the cytosol
  • the gold particles are electron dense and can be followed by microscopy
  • suggest that each pore can import and export

MCDB 428 (1 decks)

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