1
Q
What’s up with domains
A
- Domains all lie within a single polypeptide
- Not all proteins have multiple domains
- Metabolic enzymes have 1 typically
- Signalling proteins typically have multiple
- Domains help predict protein function
2
Q
what are protein chaperons
A
Protein chaperones look different, but all help a protein achieve its final form. Speed up the process, can envelope a protein, or attached to the chain
3
Q
why is whey protein a thing
A
Whey protein
- Humans cannot make all 20 amino acids
- essential amino acids must be taken up in our diet
4
Q
what’s up with vitamins and co-enzymes
A
- Enzymes and co-factors
- we cannot produce all of the vitamins required
- diet and microbes
- some enzymes require minerals
5
Q
what’s up with vitamin K
A
- Carboxyglutamate localizes factors to the wound site via interaction with calcium
- Warfarin and dicoumarol inhibit this reaction, decreases clotting. Warfarin is also a rat poison that causes them to bleed internally
6
Q
why is Mg 2+ important
A
Mg 2+ is required by dna and dna polymerase
7
Q
what are the 7 functions of nucleic acids
A
- Nucleic acids involve the nucleotides and their polymers the polynucleotides DNA and RNA
- DNA →(transcription)→ RNA →(translation)→ Protein
- information storage and retrieval
- the linear sequence of nucleotides in polynucleotides dictates the primary structure of proteins, performs many codes in its own expressions and also encodes many non protein products
- central dogma of biology
- Carriers of chemical energy
- ATP is a nucleotide. Hydrolysis of the phosphoanhydride bonds in ATP is a universal mechanism for releasing the energy required by many catalyzed reactions
- CoA is a common carrier of 2-carbon groups
- NADH and NAHPH are common carriers of electrons
- Cell signalling
- cAMP and cGMP are common cytosolic “second messengers” in signal transduction cascades
8
Q
What are Nucleotides
A
Nucleotides are composed of
- a nitrogenous base
- a pentose (5-carbon) sugar and
- one or more phosphate groups (PO4 2-)
An individual nucleotide is a nucleic acid molecule
9
Q
what are nucleosides
A
Nucleosides are not nucleic acids as they lack an acidic phosphate group
10
Q
what is the difference between ribose and deoxyribose
A
Ribose has an OH on carbon number 2, while Deoxyribose has two Hs on the same carbon
11
Q
what do each part of ATP make
A
Ribose + Adenine = Adenosine
Ribose + adenine + phosphate group = Adenosine monophosphate (AMP)
Ribose + adenine + two phosphate groups = Adenosine diphosphate (ADP)
Ribose + adenine + three phosphate groups = Adenosine triphosphate (ATP)
12
Q
what are nucleosides and nucleotides
A
Sugar + base → Nucleoside
Sugar + base + phosphate → nucleotide
13
Q
what are the two types of P-O bond
A
P-O bonds between phosphate groups = Phosphoanhydride bonds (can be broken to release lots of energy)
P-O bond that attaches phosphatees to ribose = phosphoester bond
14
Q
what is Triphosphate vs Trisphosphate
A
Triphosphate= three phosphate in a row
Trisphosphate = Phosphates make T shape with ribose
15
Q
What are polynucleotides
A
Polynucleotides are also Nucleic acids
Polynucleotides are made of many nucleotides covalently linked by phosphodiester bonds that connect 3’ to 5’ carbon
Some are single stranded (ss) such as ssDNA or ssRNA
others are double stranded (ds) such as dsDNA or dsRNA
16
Q
what are the components of DNA and RNA
A
Components of Nucleic acids
Purines = 2 rings = Adenine (A) and Guanine (G)
Pyrimidines = one ring = Thymine (T), Uracil (U), and Cytosine (C).
Thymine has an H3C in place of the H uracil has
17
Q
How was the structure of DNA discovered
A
- Nobel prize (physiology or Medicine) awarded to Francis Crick, James Watson, and Maurice Wilkins in 1962
- Rosalind Frankin grew crystals with DNA and took X ray crystallography image of it. Helped the award winners find that DNA had two strands. Not acknowledged
x ray crystallography: grow something in a crystal lattice, shoot with x rays, some level of the lattice will deflect the x ray. Complex math can be used to figure out the structure of the substance in the crystal. Used to determine structure of DNA.
18
Q
how is DNA made
A
DNA is made from a sugar phosphate backbone with planar bases lying flat like rungs on a ladder
19
Q
what misconception did people early on have about DNA
A
Early on people thought DNA would need at least 3 strands so that the bases could be accessed from the outside, but they were wrong
20
Q
what are the bonds in DNA
A
Hydrogen bonds hold the bases together. 2 between A and T/U, and 3 between C and G
Van der waals interactions cause stacking of the bases.
21
Q
what’s up with grooves in DNA
A
The space between the backbone causes 2 grooves. A bigger “major” groove and a smaller “minor” groove.
Major groove allows the nitrogenous bases to be accessed from the outside.
22
Q
what did Florence Bell and William Astbury do
A
In 1938 Florence Bell working for William Astbury came very close to discovering the structure of DNA, but only represented it with one strand.
23
Q
what are the three types of DNA
A
B DNA = most common (right handed), major and minor grooves. 10.5 bases per turn
A DNA = right hand, different grooves from B. Shorter, wider, 11 bases per turn. Bases more tilted, axis goes through the major groove.
Z DNA = left handed
24
Q
What is Watson Crick pairing
A
A with T/U and G with C = Canonical = Watson Crick pairing
Antiparallel means one strand is flipped relative to the other, both run 5’ to 3’ just in opposite directions
This explains chargaff’s rule where the ration of A to T, and G to C both equal 1. Wile G/A and T/C are different
The also led them to believe there would be a possible copying mechanism
25
what's the dogma
1. Transcription. Nuclear DNA directs the synthesis of specific mRNA molecules
2. mRNA export. mRNAs exit through nuclear pores and bind to cytoplasmic ribosomes.
3. Translation. A ribosome synthesizes the specific protein encoded by the mRNA.
26
what's transcription
DNA serves as the template to write the mRNA (messenger RNA)
Carried out by RNA polymerase (RNAP)
27
what's genetic code
The mRNA is read in triplet codons (3 nucleotides) with different combinations read as different amino acids.
There is redundancy, since there are 64 possible combinations, but only 20 amino acids
28
what is translation
mRAN is the template for protein translation
translation carried out by ribosomes
29
what is up with gene architecture
Upstream of transcriptional start site there are sequences in the DNA that affect transcription rates
All cells (in an organism) have the same genes, what genes are active determines what type of cell it is and what it does. Ex Skin, nerve, muscle, etc
The coding strand is the strand that is coped into RNA. It has the same sequence of the mRNA
Upstream = towards 5’ on coding strand. Towards 3’ on template
Downstream = the opposite
You can count residues downstream as +1, +2, +3, etc. Going upstream it is negative, -1, -2, -3, etc
30
what is up with gene regulation
A promoter region is where the transcription starts
an enhancer is a site where activators bind, which increases transcription
repressors can bind to silencers to decrease transcription
Both enhancer and silencer sites are up stream of the promoter
31
how does gene regulation affect gene expression
When there is no activator or repressor a gene is transcribed at low (basal) levels
when there is an activator, a gene is transcribed at high levels (”on”)
When a repressor is present the gene is not transcribed (”off”)
These lead to cells being different
32
what is GFP
Green Fluorescent protein (GFP) was initially isolated from a species of jellyfish
in the jellyfish a series of activators and repressors cause expression of a gene that makes GFP, which makes jelly fish glow
33
How can we use GFP
The gene for GFP can be added to replace the typically present gene at the end of a promoter region.
When the series of promoters activates the gene at a high level, the cells will become green.
If the gene is not transcribed it won’t be green
If transcribed at a basal level, it can be slightly green
34
why is GFP important
This allows us to determine how much of a particular gene is being expressed by replacing it with GFP.
GFP = a reporter gene
The Nobel prize in chemistry in 2008 was given to Osamu Shimemura, Martin Chalfic, and Roger Y Tsien for figuring out this use of GFP.
35
what are Fluorescent organisms
GFP can be added to epithelial cells to make glowing organisms.
A whole pallet of fluorescent proteins have been found and developed.
36
What are the functions of carbohydrates
Simple sugars and their polymers
Main functions
- energy storage: storage polysaccharides
Fuel molecules
- Monosaccharides
Structural support
- storage polysaccharides
Molecular recognition
- parts of receptors for extracellular signalling molecules
37
what are monosaccharides
- Major cell nutrients (esp glucose)
- their carbon skeletons are often the raw material used for building other organic molecules
- Generalized molecular formula: (CH2O)n
- Classified by two features
- number of C atoms: many structural isomers within each group
- Chemical nature of carbonyl group: aldoses are aldehydes, ketoses are ketones
38
what are the types of monosaccharides
Classification of monosaccharides based on their number of carbon atoms:
3C = trioses
4C = tetroses
5C = pentoses
6C = hexoses
7C = heptoses
8C = octoses
39
what are aldoses and ketoses
Aldoses are aldehydes: have a R-C=O -H
Ketoses are ketones: have a R-C=O -R
40
how can rings form
If a sugar has 4 or more carbons it will almost always form a ring
The ring will always attach to carbon 1 or 2
41
what is starch
Starch is the principle storage polysaccharide of plants
digested by amylases
a mixture of very long helical glucose polymers
2 principle forms: amylose and amylopectin
Amylose is a single chain of glucose monomers. Connected by 1-4 carbons
Amylopectin is brancing by 1-6 carbon linkages
42
what is glycogen
Glycogen is the principle storage polysaccharide of animals
similar to amylopectin, but branch points are more frequent
43
what is cellulose
Cellulose provides function, structure and physical support
Cellulose is the principal structural component of plant cell walls
linear unbranched polymer of glucose
not helical
broken down by cellulase
44
What are glycolipids and glycoproteins
Glycolipids are sugars attached to lipids
Glycoproteins are sugars linked to proteins
An O linked to carbohydrate is when there is a glycoside bond with a serine or threonine an H linked glycoprotein is when the ammonia carbon is linked to the amino group of asparagine
Can have branched carbohydrates attached
45
What are lipids
Diverse group of generally hydrophobic, water-insoluble organic molecules
Defining characteristics: more soluble in non-polar solvents than in polar solvents such as water
Examples:
- fatty acids
- monoglycerides, diglycerides, triglycerides
- phospholipids, glycolipids
- steroids, sterols,
- waxes
46
What are the 6 lipid functions
Energy storage
- very compact fuel reserve
- gm for gram fats have ~2x as much energy as polysaccharides
Fuel molecules
- fatty acids (released from fats) are oxidized in mitochondria
- used to form acetyl-CoA for krebs cycle
Membrane formation
- phospholipids and glyclolipids spontaneously self-seal into bilayers in aqueous solutions
Communication (cell signalling)
- steroid hormones
- some second messengers
protection of organs
- adipose tissue cushions many organs
Therm insulation
- adipose tissue decreases thermal conductivity of body coverings
47
what are fatty acids
Compounds with a hydrophilic carboxyl group and an unbranched hydrophobic hydrocarbon chain
Amphipathic or Amphiphilic: molecules that posses both polar (hydrophilic) and non-polar (hydrophobic) domains
Saturated have no double bonds
Unsaturated have at least one double bond
48
how can we represent fatty acids
Can be represented by a ration of # of carbons:# of double bonds
ex: Palmitic acid has 16:0, stearic acid has 18:0
Unsaturated have 1+ double bonds, poly unsaturated have 2+ double bonds.
Oleic acid 18:1, Linoleic acid has 18:2
49
what are Several key features contribute to structural variation in fatty acids
- length (# of carbon atoms)
- Saturated, monounsaturated, or polyunsaturated
- If unsaturated, location(s) or C-C double bonds
- if unsaturated, cis vs trans
- presence of other groups … rings, -OH groups, -CH3 groups (branches)
50
how do we count carbons
C1 is on the COOH group, count out from there
When naming, write if its cis or trans then ∆C#
ex oleic acid cis-∆9
A CH2 always separates double bonds
w (omega) carbons numbering works from the furthest carbon from the COOH inwards, w, w2, w3, etc
51
what are types of lipids
Fatty acids undergo esterification onto glycerol to form monoglycerides, diglycerides, and triglycerids
Monoglycerides = Monoacyl glycerol (MAG)
DIglycerides = DiAcylglycerol (DAG)
Triglycerides =TriAcyl glycerol (TAGs)
52
what is the structure of phosphoglyceride
Phosphoglyceride structure
Diglyceride + phosphate group = Phosphatidic acid
Diglyceride + phosphate group + Head group = Phosphoglyceride
53
what are the head groups pf phospholipids
Serine
Ethanolamine
Choline
Inositol
54
what are phospholipids
Have a phosphate, with 4 oxygens
Include phosphoglycerides (ex phosphatidylcholine) and phosphosphingolipids (ex Sphingomyelin)
Phosphoglycerides are made on a glycerol backbone
Choline + Phosphate + glycerol + 2 Fatty acids = Phosphoglycerides
Choline + phosphate + Sphingosine + fatty acid = phosphosphingolipids
55
what are two types of lipids important for neurology
Cerebrosides have no charge
Gangliosides have a charge (negative)
important for neurology
56
what are sterols and steroids
Sterols four plate like rings and a head group. Ex cholesterol which serves as a fluidity buffer
Steroid hormones derived from Cholesterol
Ex: Estradiol an estrogen
Testosterone an androgen
Cortisol a glucocorticoid
Aldosterone a mineralocorticoid
57
What shapes do phospholipids make
Can spontaneously form self-sealing bi-molecular sheets when placed in aqueous solution
Micelle are simple rings with the tails pointed inside.
Inverted micelles are simple sings with the tails pointed outside
Lipid bilayers are two layers of phospholipids with the tails pointed inward, when this forms a circle it makes a vesicle.
58
what does the membrane do
- define boundaries
- protein localization
- transport
- role in signalling
- cell to cell interactions
59
what happened with membrane research in the 1890s
1890s - Overton found that non polar materials could easily enter the cell while polar ones could not
60
what happened with membrane research in the 1900s
1900s - Langmuir used benzene to show that after it evaporated a monolayer of lipids was left on the surface of the water
61
what happened with membrane research in the 1920s
1920s - Garter and Grendel found that the surface area of Langmuirs monolayers was twice the surface area of the cells used. So the cell must have a bilayer
62
what happened with membrane research in the 1930s
1930s - Davson and Danielli theorized that the lipid bilayer would be sandwiched between two layers of protein (not correct)
63
what happened with membrane research in the 1950s
1950s - Robertson discovered the unit membrane by looking at the membrane with an e- microscope. Found there was not enough protein to fully coat the lipids (plus lipid eating techniques still affected the membrane)
64
what happened with membrane research in the 1970s
1970s - Singer and Nicolson presented the fluid mosaic membrane where proteins were embedded in the lipid bilayer
1970s - Unwin and Henderson analyzed the structure of the first membrane protein
65
What is up with the fluid mosaic model
When proteins are embedded in the membrane, the parts inside the bilayer are hydrophobic
Lipids can move laterally and spin on their axis, but not flip between leaflets (can be transported using energy)
Most lipids are unequally distributed (ex glycolipids are non cytosolic)
Phosphatidylserine on inner leaflet (dies if on outside)
Dynamics of fluidity
66
what does analysis of lipid composition
Analysis of lipid compositions demonstrates differences between membranes.
Plasma membranes from rat livers are different from those of potatoes, or e. coli, or the myeline of humans, or chloroplast thylakoids, or the inner mitochondria from rat livers.
67
What's up with FRAP
cell surface was labelled with fluorescent dye
Laser beam was used to bleach an area of the cell surface.
Fluorescent labeled molecules diffused into the bleached area. Showed the membrane was fluid.
FRAP - fluorescence recovery after photobleaching
can be used to measure recovery of lipids, proteins, or both
68
What affects membrane fluidity
Membrane fluidity can be controlled by types of fatty acid in the membrane
More unsaturated fatty acids = more fluid
More saturated = less fluid
This is because the saturated tails line up well have have lost of van der waals interactions compared to unsaturated
Raising temperatures changes it to change from gel to more liquid like
The longer lipids are the more van der waals interactions they have, the higher the melting point (= less fluid)
69
what do biological membranes need with fluidity
Biological membranes need a middle zone of fluidity where they aren’t so fluid as to fall apart, but fluid enough to fill holes
Lipid bilayer are much more disorganized then often represented
70
what is up with membrane proteins
proteins do things in the membrane
integral proteins are directly embedded in the membrane
peripheral proteins are associated with the membrane or with proteins embedded in it
Some proteins are anchored in the membrane to the cytoskeleton
they can also associated with organelles like the ER.
71
how permeable are different molecules
Hydrophobic molecules can easily pass through the membrane (such as O2, CO2, N2, steroids, and hormones)
Small uncharged polar molecules can somewhat pass through (such as H2O, urea, and glycerol) but are often
Large uncharged polar molecules are mostly prevent from crossing, but can sometimes slip by (glucose, sucrose)
Ions cannot pass, such as H+, Na+, HCO3-, K+, Ca2+, Cl-, Mg2+
72
what is simple diffusion
unassisted movement of solute molecules from an area of high concentration to lower concentration.
73
what is osmosis
membrane is permeable to solvent, but not solutes
solvent moves form area of high concentration (low concentration of solute) to low solvent concentration (high concentration of solute)
74
how do cells respond to osmosis
Animal cells get shriveled in hypertonic solutions
They are normal in isotonic solution solutions
And lysed in hypotonic solutions
Plant cells are plasmolyzed in hypertonic solutions, Flaccid in isotonic, and Turgid in hypotonic.
75
what is up with simple diffusion
Limited to small, nonpolar molecules
ex gases in and out of erythrocytes
size, polarity, charge
76
what is up with facilitated diffusion
most substances cannot diffuse across the membrane at a reasonable rate
require transport proteins
still down a concentration gradient
While simple diffusion follows a linear relationship between rate of diffusion and solute concentration. Facilitated diffusion follows a hyperbolic relationship since it requires a protein to happen, so this limits it later on.
77
What are the two types of transporting proteins, how do they work
Transporters = carrier proteins
Channel proteins = pores in membrane with polar residues along the edges to allow diffusion through it
Carrier proteins act like airlocks, only one side is open at a time. A solute will enter on one side, bind to a solute-binding site, the carrier will undergo evertion (it will evert) to close one side and open the other. Evertion is energetically favourable
78
what's up with carrier proteins
Carrier proteins:
- sometimes called permeases
- highly specific
- transport velocity has an upper limit
- transport one or two solutes
79
What's up with GLUT1
1. Glucose binds to a GLUT1 transporter protein that has its binding site open to the outside of the cell (T1 conformation)
2. Glucose binding causes the GLUT1 transported to shift to its T2 conformation with the binding site open to the inside of the cell
3. Glucose is released to the interior of the cell, initiating a second conformational change in GLUT1
4. Loss of bound glucose causes GLUT1 to return to its original T1 conformation, ready for a further transport cycle
Can work in other direction depending on concentration
inside the cell a phosphate group is added to glucose, this causes GLUT1 to not recognize it, keeping it inside the cell
80
what's up with channel proteins
Form hydrophilic channels
ex Ion channels
may display high degree of selectivity
may be selectively opened and closed (gated) but diffusion is passive
81
How does cystic fibrosis happen
In a normal cell, CFTR protein pumps Cl- into the airway lumen mucus lining. Once Cl- is present, it pulls Na+ between the cells into the mucus. Now that the mucus has salt, it pulls water in to keep it hydrates.
In cells with cystic fibrosis, the CFTR protein is absent/dysfunctional. No Cl- entres the airway, no Na+ or water are pulled after it. The mucus is dehydrated and infected with bacteria
82
what are porins
Porins form Beta-Barrel hydrophilic channels
aquaporins contain transmembrane helices
Beta sheats wrapped around into a cylinder, hydrophobic R groups on outside, hydrophilic on inside.
83
what is active transport
moving molecules up a concentration gradient is active transport
this requires an input of energy
can be separated based on source of energy input
84
what are the two types of active transport
Direct active transport: linked to exergonic chemical reaction (often ATP hydrolysis)
Indirect active transport: utilizes existing gradients to drive transport. (ex exergonic inward movement of protons provides energy to move the solute up its concentration gradient)
85
What are P type ATPases
P = phosphoryl group
transporter is temporarily and reversibly phosphorylated during transport
humans have about 70 P-type ATPase pumps
1. Three Na+ from inside bing to E1
2. Na+ binding triggers autophosphorylation of the alpha subunit using ATP and ADP is released
3. A conformational change to E2 expels three Na+ to the outside of the cell
4. two K+ from outside the cell bind to E2
5. K+ binding triggers dephosphorylation causing a conformational change back to E1
6. Two K+ are expelled to the inside as ATP binds and pump returns to initial state
86
what are V type ATPases
V = vacuole
these pump protons into organellar compartments (remember lysosomes)
Pump is not phosphorylated in the process
H+ pump keeps pH difference of 5.0 inside vs 7.2 outside lysosomes
87
what are Acid hydrolases
Acid hydrolases:
- nucleases
- proteases
- glycosidases
- lipases
- phosphatases
- sulfatases
- phospholipases
88
what are F type ATPases
F = factor
two components, Fo which is a transmembrane pore for protons, and F1 which include ATP binding site
can either pump protons or make ATP. In normal cellular conditions, uses H+ movement to make ATP
89
describe the Fo static component
The Fo static component consists of one a and two b subunits. The a subunit forms the proton channel and is immobilized in the membrane. The b subunit forms the peripheral stalk and are attached to both the a subunit and F1
90
describe the Fo mobile component
The Fo mobile component consists of a ring of 10 c subunits. Only one c subunit can form an ionic bond with the a subunit at a time. For each proton translocated the ring rotates one tenth of a turn as the adjacent c subunit in the ring bonds with the a subunit.
91
describe the F1 static component
The F1 static component consists of the δ (delta) subunit plus a catalytic ring formed by a hexagon of alternating α (alpha) and β (beta) subunits. The α3β3 ring is the site of ATP synthesis and is immobilized by the δ subunit, which connects it to the b2 peripheral stalk of Fo.
92
describe the F1 mobile component
The F1 mobile component consists of the ε (Epsilon) and γ (gamma) subunits, which form the central stalk that is firmly attached to the c10 ring of Fo. As proton translocation turns the C10 ring, the γ subunit rotates inside the α3β3 catalytic ring of F1.
93
what are ABC transporters
Each protein contains 4 domains
- 2 ATP binding cassettes
- ABCs
- 2 membrane spanning
At least 70 ABC transporters in the human genome
Important role in multi drug resistance of cancers
CFTR is a type of ABC transporter (the transporter that is defective in cystic fibrosis)
94
What is indirect active transport
Links energetically favourable transport with energetically unfavourable
this means one solute is moving down a concentration gradient while the other moves up a gradient
Utilize symporters and antiporters
Symporters have both solutes moving the same direction
Antiporters have both moving different directions
95
describe the Na+ glucose symporter
1. Two sodium ions from outside the cell are bound
2. Binding of sodium ions allows glucose binding and a subsequent conformational change
3. Symporter opens to inside
4. Sodium ions are released inside, but are continually extruded to outside by a separate sodium-potassium pump
5. Loss of sodium ions is followed by glucose release to inside
6. Release of glucose allows the empty symporter to return to initial state
96
describe Bacteriorhodopsin
Proton pump
retinal embedded in the protein
retinal changes from all trans to a form with one cis double bond
used by bacteria and archae
ex: Halobacterium salinarum
Not dependent on ETC
light-driven proton pump
Bio 225
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