Exam 1 Flashcards
(270 cards)
1
Q
What size are most tissue culture cells?
A
20-30 um
2
Q
What sizes can light microscopy see?
A
from 25 nm to 10 mm
3
Q
What kind of organism and structures can you see with light microscopy?
A
- frog eggs
- plant cell
- animal cell
- bacteria
- viral ribosomes
4
Q
What is the smallest organisim/structure that the conventional resolution of light microscopy can show?
A
- bacteria
5
Q
What is the smallest organisms/structure that super resolution of light microscopy?
A
viral ribosomes
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
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
8
Q
What is a major challenge in transmission microscopy?
A
- maintaining or introducing contrast
9
Q
What does the hematoxylin stain do?
A
- basic dyes the nucleus
10
Q
What does the eosin stain do?
A
- acidic dyes that stains the cytosol
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
12
Q
What does the first barrier filter do?
A
- lets only blue light through with a wavelength between 450-490nm
13
Q
What does the beam-splitting mirror do?
A
- reflects light below 510 nm and transmits light above 510nm
14
Q
What does the second barrier filter do?
A
- cuts out unwanted fluorescent signals, passing specific green fluorescent emission between 520-560nm
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
16
Q
What does DAPI stain for?
A
- DNA/the nucleus
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)
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
19
Q
What is GFP used for?
A
- used in live cell imaging once fused to organisms’ DNA
20
Q
What can you use to study membrane protein degradation in live cells?
A
dual color staining/imaging
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
22
Q
What is FRAP and FLIP?
A
- FRAP = Fluorescence Recovery after Photobleach
- FLIP = Fluorescence Loss in photobleaching
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
24
Q
What is the physical limit of resolution for a light microscope?
A
~0.2um
25
Why is there a resolution limit on a light microscope?
- visible light is 380-750nm (.38-.75um)
- need to increase resolution to go lower
26
What is the resolution of a super resolution microscope?
~30-50nm
27
What is the resolution of an electron microscope?
0.1nm
28
What is the diffraction limit of a normal microscope?
- 1/2 of the wavelength (on the x-y plane)
29
What is diffraction limit?
- the maximum resolution
30
What are examples of super-resolution microscopies?
- Structured Illumination microscopy (SIM)
- Photo activated localization microscopy (PALM)
- Stochastic Optical Reconstruction Microscopy (STORM)
- Stimulated Emission Depletion (STED)
31
What does STORM do to fluorescence?
- makes the lights less blurred and more pinpoint
32
How do PALM and STORM techniques work?
- 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
33
What is transmission electron microscopy (TEM)?
- 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
34
What are the advantages of EM?
- high resolution (down to 0.1nm)
- can see ribosomes and large protein complexes
- can see membrane bilayer (~5nm wide)
35
What are the disadvantages of EM?
- 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
36
How does immune-gold labeling work for EM?
- 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
37
What is Scanning electron microscopy (SEM)?
- collects reflected electrons off the surface of samples
- based on what angle they are reflected, creates images
38
What part does SEM and TEM visualize?
- TEM can only see a cross section of a cell/structures
- SEM can only see surfaces not inside structures
39
What is freeze-fracturing used for?
visualize the surfaces of internal structures
40
What is the process of freeze-fracturing?
- 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
41
What is the process of monoclonal antibody production?
- 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
42
What is velocity sedimentation?
- separates particles based on their relative size M(r)
- an example of this is cell fractionation
43
What is M(r)?
- molecular radius
- a combination of both size and density
44
What is the process of velocity sedimentation?
- 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
45
What is differential centrifugation?
- uses increasingly faster speeds to separate different particle groups
46
What structures do you receive after low-speed centrifugation (1000 x g, 10min)?
larger structures:
- whole cells
- nuclei
- cytoskeletons
47
What structures do you get after medium-speed centrifugation (20,000 x g, 20min)?
smaller organelles:
- mitochondria
- lysosomes
- peroxisomes
48
What structures do you get after high-speed centrifugation (80,000 x g, 1hr)?
- microsomes
- small vesicles
49
What structures do you get after very high-speed centrifugation (150,000 x g, 3hr)?
- ribosomes
- viruses
- large macromolecules
50
What is an equilibrium gradient?
separates structures of different densities, not size
51
How does the protein composition of different organelles affect the results of doing an equilibrium gradient?
- the presence of distinct proteins in each organelle increases their densities differently
52
How does an equilibrium gradient work?
- 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
53
What does SDS-page gel do?
separates proteins based on their sizes
- linearizes the protein
54
How do antibodies function in a western blot?
- 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
55
What is the purpose of a pulse-chase experiment?
- to study the life span, trafficking, modification and death of a protein
56
What is the pulse phase in a pulse-chase experiment?
- chemically tag newly synthesized proteins
- tag is to metabolically label with radioactive amino acids
57
What is the chase phase in a pulse-chase experiment?
- 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
58
What is forward genetics?
- 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
59
What is reverse genetics?
- finding a gene of interest and then mutating the gene and finding a phenotype
60
What is the myostatin gene?
- a gene that regulates muscle development and mass
- when mutated, you get an abundance of muscle mass
61
How can conditional mutations be used to study essential genes?
- 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
62
What does a complementation analysis do?
- to see if 2 mutations that produce the same phenotype are on the same gene
63
How does a complementation analysis work?
- 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
64
What is the point of RNAi/siRNA?
- 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.
65
How does an RNAi experiment work?
- 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
66
What does CRISPR/Cas9 do?
- adapted from bacteria to help edit genomes
- causes dsDNA breaks
- can repress or activate gene expression
67
How does the CRISPR/Cas9 system work?
- 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)
68
What are the characteristics of proteins?
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
69
What is the secondary structure of proteins
- regular repeating arrangement of backbone + hydrogen bonds
- 3D segments called domains
70
What is protein tertiary structure?
- 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
71
What is protein quaternary structure?
- fitting multiple polypeptides together
- association of multiple polypeptides to form complexes
72
What is macromolecular assembly?
- regulated assembly of proteins, large complex or protein machine
73
Is cysteine polar or non polar?
- nonpolar
74
What are characteristics of hydrophobic amino acids?
- tend to stay on the inside of folded proteins to keep away from the hydrophilic environment
- form transmembrane domains to span lipid bilayers
75
What is special about the hydrophobic tyrosine amino acid?
- contains a hydroxyl group on its benzyl ring
- can be phosphorylated during signaling
76
What are the characteristics of Alanine?
- Ala
- A
- non polar/hydrophobic
77
What are the characteristics of Glycine?
- Gly
- G
- non polar/hydrophobic
78
What are the characteristics of Valine?
- Val
- V
non polar/hydrophobic
79
What are the characteristics of Leucine?
- Leu
- L
- non polar/hydrophobic
80
What are the characteristics of Isoleucine?
- Ile
- I
non polar/hydrophobic
81
What are the characteristics of Proline
- Pro
- P
nonpolar/hydrophobic
82
What are the characteristics of Phenylalanine?
- Phe
- F
non polar/hydrophobic
83
What are the characteristics of Methionine?
- Met
- M
nonpolar/hydrophobic
84
What are the characteristics of Tryptophan?
- Trp
- W
nonpolar/hydrophobic
85
What are the characteristics of Cysteine?
- Cys
- C
nonpolar/hydrophobic?
86
What are the characteristics of Aspartic acid?
- Asp
- D
negatively charged
87
What are the characteristics of glutamic acid?
- Glu
- E
negatively charged
88
What are the characteristics of Arginine?
- Arg
- R
Positively charged
89
What are the characteristics of Lysine?
- Lys
- K
positively charged
90
What are the characteristics of Histidine?
- His
- H
positively charged
91
What are the characteristics of Asparagine?
- Asn
- N
polar
92
What are the characteristics of Glutamine?
- Gln
- Q
polar
93
What are the characteristics of Serine?
- Ser
- S
polar
94
What are the characteristics of Threonine?
- Thr
- T
polar
95
What are the characteristics of tyrosine?
- Tyr
- Y
polar
96
What are the characteristics of hydrophilic Amino acids?
- 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
97
What is special about Cysteine?
- can form intra or inter-molecular disulfide bonds
- site of post-translational fatty acylation (prenylation. palmitoylation, farnesylation) of membrane associated proteins
98
What is special about glycine?
- has a hydrogen as its side group
- this makes it packs well and often to save as a flexible helix breaking residue
99
What is special about proline?
- 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
100
What is special about aromatic AAs?
- can be used to calculate protein concentration
- Trp rings absorb the most UV, then tyrosine, then phe
101
What amino acids can be phosphorylated?
- ser, thr and tyr
102
What AAs can be glycosylated?
- 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
103
What is the MW of an amino acid?
- 110 Daltons / 110 grams per mole
104
What are the characteristics of polypeptides?
- 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
105
What are the non covalent bonds that help with protein folding?
- electrostatic attractions
- hydrogen bonds
- van der Waals attractions
106
Why are some 3D structures stable?
- hydrogen bonding of backbones amide and the carbonyl groups
107
T/F: secondary structures does not generally depend on the types of amino acids to comprise structure
true
108
What are the 3 main secondary structures for proteins?
- alpha helices
- beta sheets
- beta turns
* proteins fold into a conformation of the lowest energy
109
How many degrees does each amino acid turn?
100
110
How many amino acids are in every turn of a helix?
3.6
111
What is the distance between amino acids 1 full turn and on the same face of a helix?
0.54nm
112
Where does hydrogen bonding occur to stabilize and alpha helix?
- between the N-H and C=O of peptide bonds 4 residues apart
113
How many AAs are typically in 1 a helix?
7 = two full turns
114
How are a helices arranged?
- side. chains are pointed out.
- can be amphipathic by one side being hydrophobic, and another side is hydrophilic
115
What interactions do a helices take part in normally?
- protein-protein
- are usually what consists of transmembrane-spanning domains (18-22AAs long when no proline present)
116
What happens if there is a proline or glycine present in an a helix?
- it would break into 2 helices
117
How many AAs in a strand of a beta sheet?
- 5-8 residues
118
Where does hydrogen bonding occur in beta sheets?
- occurs between strands, not adjacent AAs
- side chains stick out above and below strands
119
How are the strands arranged?
can be either parallel or anti-parallel
120
What is circular dichroism?
- the way left circular and right circular polarized lights are absorbed differently by different secondary structures
121
How is circular dichroism in protein secondary structure analysis?
- measuring absorbances of the different light polarities can be used to estimate the content of secondary structures
122
What are the 3 main classes of tertiary structure?
- globular
- fibrous
- transmembrane
123
What is a structural motif?
- 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
What is a structural domain?
-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
What is a coiled coil?
- 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
What is an EF-hand domain?
- 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
What is a Zn-finger domain?
- DNA binding domain
- zinc binds, coordinated by two pairs of His and Cys residues
128
What is a structural domain?
- combination of secondary structures used for a specific kind of function
-distinct region of a tertiary structure
129
What has been developed and used over recent years to predict protein folding with >90% accuracy?
- AI (alpha fold)
130
What are the main roles of membranes?
- separate all cellular processes from the outside environment
- maintain compartmentalization of different organelles
131
What constitutes a membrane?
50% lipids + 50% proteins
- molar ratio 50:1
132
What are the 3 main types of lipids?
- phosphoglycerides (PC,PE, PS)
- sphingolipids
- sterols
* phosphinositides are less abundant but prevalent to signaling
** also glycolipids
133
What are the characteristics of phosphoglycerides?
- 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
What are the characteristics of PE?
phosphatidylethanolamine
-phosphoglyceride
- has an ethanol amine group
- net neutral charge
(negative charge of phosphate group + positive charge on the amine group cancel out)
135
What are the characteristics of PS?
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
What are the characteristics of PC?
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
What are the characteristics of the sphingosine backbone?
- 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
What are the characteristics of sphingomyelin?
- 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
What 4 types of lipids compose a majority of the mammalian membrane?
- PE
- PS
- PC
- SM
140
What is the common structure of most sterol?
- a 4 ring hydrocarbon group
141
What are the characteristics of cholesterol?
- 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
What are the common characteristics of glycolipids?
- 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
What is galactocerebroside?
- glycolipid
- major in mammalian lipid bilayers
- sphingosine backbone
- has a galactose group attached
144
What is ganglioside?
- glycolipid
- sphingosine backbone
- has 5 sugar groups attached at the third carbon
- negatively charged NANA group
145
What are common characteristics of phosphoinositides?
- 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
T/F: most lipids are amphipathic
true
147
What structures do lipids normally form into?
- 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
How do lipids move most commonly?
- laterally diffuse the most
- fluidity depends on its composition and temperature
- flipping across layers rarely occurs
149
What happens with the membrane at lower temperatures?
- 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
T/F: transmembrane proteins can't laterally diffuse within a membrane
false: they can
151
What enzyme allows for lipids to flip between bilayers of membranes?
flippases
152
True or False; the lipid bilayers are asymmetrically composed of different lipid types.
true
153
What types of lipids are prevalent on the extracellular layer of the plasma membrane?
- Phosphatidylcholine
- sphingomyelin
- glycolipids
- typically the heavier layer is oil the outside membrane
154
What types of lipids are prevalent on the cytosolic layer of the plasma membrane?
- Phosphatidylethanolamine
- Phosphatidylserine
- phosphoinositides
- phosphoinositide phosphates
155
What side of the plasma membrane would the lumen side of a transmembrane protein face?
- extracellular face
156
T/F: both lipids and proteins can be glycosylated
true
157
What forms a heavy layer on the outer membrane to form a protective barrier around the cell?
- glycosylation of membrane proteins, lipids, and carbohydrates
158
Where do lipids come from?
- the ER
- packaged and transported in and from the golgi
159
Where are the enzymes that glycosylate proteins and lipids?
- ER lumen
160
Why would it be important for PIPs (Phosphoinositides phosphates) to be on the cytosolic side of the plasma membrane?
- to be easily accessible for their major role as signaling molecules
161
What are the types/ways that different proteins associate with membranes?
- 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
How can peripheral proteins be extracted?
- with a solution that has a pH >10 or high pH salt
163
How can GPI-anchored proteins be extracted?
- released by using phospholipase C (PLC)
164
how can integral membrane proteins be extracted from the plasma membrane?
- can only be released with detergents
165
What are the characteristics of a hydrophobic alpha helices region of a protein on a hydropathy plot?
- has a peak in the positive area
- is >18 amino acids long
166
What determines the different types of a lipid anchor?
- how many carbons are in the fatty acid chain
- where on a protein is the anchor attached
167
What are the characteristics of myristoylation/myristoyl anchors?
- 14C fatty acid chain
- attached to the first glycine on the N-terminus end of a protein
168
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?
- prior amino acids are cleaved off
169
What are the characteristics of palmitoylation anchors?
- 16C fatty acid chain
- attaches to a cysteine on the protein
- thioester linkage
170
What are the characteristics of farnesylation anchors?
- 15C fatty acid chains
- forms a thioester bond between the prenyl group and a cysteine on the protein
171
What is the common face that myristoyl, palmitoyl and farnesyl anchors are on?
the cytosolic face
172
What are the characteristics of a GPI anchor?
- 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
What direction do GPI anchors face?
- because these are constructed and attached in the ER lumen, they always face outside of the cell
174
How can GPI-anchored proteins be released from the membrane?
- using an inositol-specific phospholipase C enzyme
175
What are all the parts of a GPI anchor?
- glycerol backbone
- phosphate group
- inositol
- glucosamine (1)
- mannose (3)
- ethanolamine
176
What is the process of reconstituting membrane proteins into liposomes?
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
What is the order of most permeable to least permeable molecules across a membrane?
- hydrophobic molecules (gases with no charge)
- small uncharged polar molecules
- large uncharged polar molecules
- ions
178
Is the Na+ concentration higher inside or outside the cell?
outside > inside
179
is the K+ concentration higher on the inside or outside of the cell?
inside > outside
180
Is the Mg2+ concentration higher on the inside or outside?
outside > inside
181
Is the Ca2+ concentration higher on the inside or outside of a cell?
outside > inside
182
Is the proton concentration higher on the inside or outside of the cell?
inside > outside
183
Is the Cl- concentration higher on the inside or outside of the cell?
outside > inside
184
What is the largest family of membrane transporters?
ABC-transporters
185
What role does membrane transport play in Cystic fibrosis and CTFR?
- doesn't allow for the adequate transport/secretion of pancreatic enzymes
186
What role does membrane transport play in bile salt transporter disorders?
- ABC transporters in the liver inadequately transport or allow the secretion of bile
187
What role does membrane transport play in retinal degeneration?
- ABCA4 transporter doesn't allow the passage retinol into the cells
188
What role does membrane transport play in mitochondrial iron homeostasis?
- ABCB7 transporter is mutated, causing anemia and ataxia
189
What role does membrane transport play in multi drug resistance?
- ABC family transporters export drugs out of the cell to prevent harmful effects
190
What are the causes/characteristics of Wilson's disease?
- 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
What are the causes/characteristics of Niemann-Pick Disease type C?
- 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
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What are the 2 main types of membrane transport?
- simple, passive diffusion
- mediated transport
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What types of molecules undergo simple diffusion?
- organic molecules
- small uncharged molecules
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What are the 2 subtypes of Mediated transport?
- passive transport
- active transport
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What is passive transport?
- facilitated diffusion and channels
- solutes go from high to low concentration
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What is active transport?
- ATP powered/energy powered pumps and transporters
- from low to high solute concentration
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Is membrane transport or diffusion faster?
- membrane transport
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What limits membrane transport?
- the amount of proteins/enzymes to mediate transport
- can become saturated and display a Vmax
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What can change the speed of diffusion?
- substrate concentration
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what are the general characteristics of membrane transporters?
- undergoes a conformational change during transportation
- relatively slow
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What are the general characteristics of channel proteins?
- a pore
- always downhill for concentration gradients
- super fast
202
What about a transporter mediates the passive movement of a solute?
conformational change of the transporter
- transition occurs randomly and are reversible
203
What is a requirement of all forms of facilitated transport?
- need a pre-formed gradient
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What are the 3 kinds of facilitated transport?
- uniport
- symport
- antiport
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What type of transporter is GLUT1?
- a glucose uniporter
- goes from high concentration outside the cell to low concentration inside the cell
-
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What is Km in relation to GLUT1?
- the concentration of glucose at which half of GLUT1 in the membrane are bound to glucose
207
What changes the conformation of GLUT1 to allow glucose to enter the cell?
- the binding of glucose in the outward-facing conformation
- can be reversed when glucose is released into the cell
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What is the Km for glucose?
~1.5mM
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Why are cytosolic levels of glucose so low and keep bringing more glucose in?
- glucose is rapidly converted to G6P in the cell, bringing down the glucose concentration
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What are the 3 main types of active transport membrane transporters?
- coupled transporters: anti port and symporter
- ATP-driven pump: ATPase
- light-driven pump
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When can the conformation change occur in a symporter?
- only when all binding sites are fully occupied or completely empty
212
In which cases would it be difficult to bring glucose into the cell?
- cytosolic glucose concentration is high
(like in small intestinal epithelium)
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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?
- 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
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What are the characteristics of the Na+-linked glucose transporter?
- glucose + 2Na+ ions are binding to the symporter (cooperative)
- causing the conformational change and release of molecules into the cytoplasm
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What makes the reverse reaction of symport of 2Na+ and glucose out of the cell?
- this reaction is still reversible
- the Low cytosolic Na+ concentration makes it less likely to occur tho
216
What are the 3 main families of ATP- pumps?
- p-type pump
- ABC transporter
- V-type and F-type pumps/synthases
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What are the characteristics of the P-type ATP pump?
- phosphorylates themselves during transport, like ATP7B
- phosphorylation and dephosphorylation powers conformational changes
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What are examples of P-type pumps?
- 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)
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What are the general characteristics of the Na+/K+ P-type ATPase?
- 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
What are the general characteristics of the Na+/K+ ATPase transport cycle?
- transient catalytic phosphorylation
- 2 conformational changes: E1 and E2
- Na+ dependent phosphorylation
- K+ dependent dephospho rylation
221
What is the transport cycle process of the Na+/K+ ATPase?
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
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What specific cell type is the sarcoplasmic reticulum found in?
a special type of ER in muscle cells
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What is the general characteristics of the SR Ca2+/ATPase pumping cycle?
- calcium dependent phosphorylation
- proton dependent dephosphorylation
- majority of the membrane proteins in the SR
224
What is the pumping cycle process of the SR Ca2+ ATPase?
- 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
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What are some common examples or cases of V-type ATP pumps?
- 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
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What is the main role of V-type pumps?
- pumps protons out of a cell across a membrane using ATP as energy
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What are common examples of F-type ATP pumps?
- bacterial plasma membrane
- inner mitochondrial membrane
- thylakoid membrane of chloroplasts
228
What is the main role of F-type ATP pumps?
synthesizes ATP by bringing in protons down its concentration gradient across a membrane
229
What are some common examples of ABC transports as ATP pumps?
- bacterial plasma membranes to transport AAs, sugars and peptides
- Mammalian plasma membranes to transport phospholipids, drugs and cholesterol
230
How does ATP binding work in the CFTR Cl- channel?
- ATP binding and hydrolysis drives the opening and closing of the Cl- channel instead of transporting ions across a membrane
231
What are the 4 types of gated ion channels?
- voltage-gated (K+ channels)
- extracellular ligand-gated (acetylcholine receptors)
- intracellular ligand-gated (IP3-gated Ca2+ channel)
- mechanically gated (membrane tension linked to cytoskeleton)
232
What are the major differences between membrane transporters and channels?
- conformation change during the passage of solutes (transporters only)
- channels are much faster than transporters
233
What do small conformation changes allow channels to/what do they change?
- 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
What are the characteristics of the K+ channel structure?
- 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
How does the pore helix help to draw in K+ ions through the filter or into the channel?
- the negative dipole end is point at the inner pore where K+ will pass through. Opposite charge draws K+ through
236
What kind of Amino acids line the membrane faces of the inner and outer helices of the K+ channel?
negatively charged AAs to draw in the positively charged K+ ions
237
What is the net charge on the cytosolic side of the cell?
negative
238
What is the net charge on the extracellular side of the cell?
positive
239
How do all excitable cells generate action potentials
voltage-gated cation channels
240
What are the 4 parts of a voltage-gated Na+ channel?
1) voltage sensors
2) central channel
3) selectivity filter
4) inactivation gate
241
What is the voltage sensor on a voltage-gate cation channel/what does it do?
- 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
What is the point of the refractory state?
-inactivation gate plugs the pore
- this stops backwards and reactivation of channels
- stays there until after the membrane is repolarized
243
When does the channel become inactivated by the inactivation gate?
- when the equilibrium potential is reached
244
What is propagation of action potentials?
- signals maintain strength by working in one direction
- channels are opened one-by-one as nearby action potentials are depolarized
245
What is the process of Na+/K+ ATPase action potential and depolarization?
- 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
What is the use of a Patch-Clamp?
- used to record individual ion channels
- you use a glass micropipette with gentle suction
- pulls about individual ion channels off of the membrane
247
What allows for an increased speed of action potentials in axons?
- 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
What is the process of transmitting a signal across a neuromuscular junction?
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
What are the two signaling pathways that translated proteins can take?
- co-translational
- post-translational
250
What is the general process of the secretory pathway in the cell?
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
Where do post-translationally targeted proteins tend to go?
- nucleus
- mitochondria
- plastids
- peroxisomes
252
Where to co-translationally targets proteins tend to go?
- plasma membrane
- tonoplasts
- secreted proteins
- cell wall
- vacuole
253
What are the 3 ways that proteins move between compartments in the cell?
- gated transport
- protein translocation across the membrane
- vesicular transport
254
What is gated protein transport?
- movement of proteins and RNA between the cytosol and nucleus
- mainly folded proteins
255
What is protein translocation across the membrane?
- proteins moves into the ER and mitochondria, chloroplast
- needs to unfold and fold proteins
256
What is vesicular protein transport?
- proteins do not cross a membrane
- mainly folded proteins
257
What parts of the cell undergo gated transport?
- between the cytosol and nucleus
258
What parts of the cell undergo protein translocation across the membrane?
Cytosol to:
- plastids
- mitochondria
- ER
- peroxisomes
259
What parts of the cell undergoes vesicular transport?
- peroxisomes
- ER
- Golgi
- Late endosome
- lysosome
- early endosome
- cell exterior
260
What is a main characteristic of nuclear pores?
- spans both membranes
- nuclear envelope is continuous with ER membrane
261
What is the role of cytosolic fibrils?
- grabs incoming cargo
262
What is the ring of the nuclear pore like?
- octagonal
- 8-fold rotational symmetry
263
What are the characteristics of gated diffusion of nuclear pore complexes?
- small molecules enter through via free diffusion
- macromolecules >60kDa have to be unfolded unless there is energy consumption to send it through
264
What are the characteristics of nuclear transport?
- 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
What is the signal sequence like when the protein is being imported into the nucleus?
- positively charged amino acids (Arg, Lys)
266
What are the signal sequences like on proteins that are being exported from the nucleus?
- leucine rich, amphipathic
- Met, leu, Phe
267
What is the nuclear localization sequence (NLS)?
- 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
What are characteristics of NLS?
- the precise sequence varies for different nuclear proteins
- can be anywhere in the proteins
- positive charge
- both necessary and sufficient for nuclear import
269
What are the characteristics/ process of the NLS-gold experiment?
- 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
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