Biochem 1 Flashcards
(165 cards)
1
Q
biochemistry
A
-study of life at the molecular level
2
Q
thermodynamics
A
-the relationship between energy, work, and heat
3
Q
energy
A
capacity to do work
4
Q
work
A
transfer of energy from the system to surroundings that can raise a weight
-organized- allows you to do something with that energy
5
Q
heat
A
transfer of energy as a result of a difference in temperature
-disorganized- difference in temp
6
Q
system
A
- what we define
- what were studying
7
Q
surroundings
A
-everything else in the universe other than the system
8
Q
does life obey the law of thermodynamics
A
- when you breakdown into smaller parts -> yes
- add up all those processes and yes it does obey
- need to work on a smaller scale first
9
Q
1st law of thermodynamics
A
- any change in the internal energy (U) of a system must equal the transfer of energy as heat or work
- energy cannot be created or destroyed
- energy of system + energy of surroundings will always = energy of universe (constant)
- Δ U= U final - U initial = q - w
- heat is released by the system and work is done by the system
10
Q
enthalpy
A
- thermodynamic potential of a system
- H
- H= U + PV
- at constant pressure enthalpy equals heat
- defined in kJ
11
Q
exothermic
A
- release of energy
- change of enthalpy is negative
- -ΔH
- release heat
- ex. -10kJ
12
Q
endothermic
A
- requires addition of heat
- change of enthalpy is positive
- +ΔH
- ex. 10kJ
13
Q
spontaneous processes
A
- have a tendency to occur without input of energy
- cracking an egg and pick up the egg and drop it again -> it wont reform bc it requires a lot of energy -> non-spontaneous
- all gas is packed into one side a chamber, when the division is lifted the gas molecules with naturally diffuse without inputting energy -> spontaneous
- ΔH < TΔS
14
Q
nonspontaneous process
A
- requires energy for process to occur
- ΔH > TΔS
15
Q
entropy (2nd law of thermodynamics)
A
- ΔS tends to increase
- disorder, randomness
- S universe is always positive (ΔS>0)
- the entropy of a system can decrease but that means the entropy of the surrounds must increase by a greater amount so that ΔS is always positive
- S
- number of energetically equivalent arrangements (J/K)
- when the partition of a chamber is lifted the gas diffuses and the entropy increases bc there are many more ways for the gas molecules to be placed -> energy will spread out within a given space
- energy moves from high to low until equilibrium (highest entropy)
- a function of temperature
- if a spontaneous process has no change in energy or enthalpy, the change in entropy must be greater than zero
- ΔS system + ΔS surroundings = ΔS universe > 0
16
Q
gibbs free energy
A
Δ S >= ΔH / T
-related entropy to enthalpy via temperature
-came up with criteria for spontaneous process:
ΔH - TΔS <= 0
-if greater than 0 its nonspontaneous and if less than it is spontaneous
Δ G = Δ H - TΔS
-if ΔG is neg its spontaneous and positive is nonspontaneous
17
Q
mechanical example gibbs free energy
A
- raising a block up a hill -> needs energy to go up the hill -> positive G -> nonspontaneous -> endergonic
- weight at the top of the hill -> doesnt require input of energy -> neg G -> spontaneous -> exergonic
18
Q
biochemical example of gibbs free energy
A
- set of reactants have free energy
- set of products that have a lower free energy
- G is negative
- exergonic reaction
- spontaneous
19
Q
exergonic
A
-if change in G is less than or equal to 0 the process will occur spontaneously
20
Q
endergonic
A
-if change in G is greater than 0 the process will not occur spontaneously
21
Q
negative H, positive S
A
- enthalpically favored and entropically favored
- spontaneous at all temperatures
- exothermic
22
Q
negative H, negative S
A
- enthalpically favored and entropically unfavored
- spontaneous at temperatures below T= Δ H / Δ S
- exothermic
23
Q
positive H, positive S
A
- enthalpically unfavored and entropically favored
- spontaneous at temperatures above T= ΔH/ΔS
- endothermic
24
Q
positive H, negative S
A
- enthalpically unfavored and entropically unfavored
- nonspontaneous at all temperatures
- endothermic
25
where does energy come from- ATP
- coupling reactions to a form of energy
- ATP
- body uses this for chemical reactions
- high energy bonds in the phosphates -> break these bonds for energy
- exergonic, spontaneous
- phosphorylate
- ex. nonspontaneous rxn -> take ATP (exergonic & spontaneous) -> couple each reaction -> **ATP reacts directly with metabolite that needs "activation" -> overall reaction has a -ΔG, exergonic
- concentration of ATP in our cells is much higher than you would expect
- the concentration of ADP will affect the free energy of the coupled chemical rxn
- the amount of energy released by converting ATP to ADP needs to be greater than the amount of energy consumed by the coupled chemical rxn
- 2 chemical rxn need to share a common intermediate to be coupled
26
concentrations of the reactions and products
- free energy change of a rxn depends on the concentrations of the reactants and products
- ex. lifting the partition in a gas chamber -> change in entropy but also a change in concentration -> closed partition (more concentration)
- change in concentration causes a change in entropy
27
standard free energy change
- constant
| - ΔG^degree
28
standard condition
- 25C
- 1 atm
- activity of water is 1
- pH 7
- reactants with multiple ionization states are considered to be in the most common state at pH 7
29
equilibrium
- the free energy change of the forward reaction exactly balances that of the reverse reaction
- ΔG is equal to zero
- we can calculate where a rxn will reach equilibrium from standard free energy data
30
Van't Hoff equation
- used to determine equilibrium constant
- tells you if rxn is spontaneous or not
- allow rxn to reach equilibrium and then measure the concentrations of reactants and products -> from that we can calculate the equilibrium constant at that temperature
- repeat this using different temperatures -> creates a linear slope trend
- plot can be used to determine if the spontaneity of a rxn will depend on temperature
- slope = -ΔH/R
- y-intercept= ΔS/R
31
positive y-intercept, positive slope
- positive slope means that ΔH is negative
- if ΔH is negative -> exothermic
- slope = -ΔH/R
32
hydrophobic effect
- 2nd law of thermodynamics (entropy)
- two nonpolar molecules in water will come together so that more water molecules will be able to freely interact with other water molecules -> increases disorder -> favored
- increases entropy when they come together
- if they were to stay apart more water molecules would be "used up" by interacting with the nonpolar molecules
33
intracellular process
- if a cell is carrying out a function it is using energy
- this energy is released as heat to the surroundings
- this increases the entropy of the surrounds
- ΔS=-ΔH/T
34
water
- primary solvent of life
- shape the biological molecules that are dissolved in it
- tendency to dissociate
- partial + and - -> permanent dipole -> polar -> allows for hydrogen bonds
- dissociates into H+ and -OH
- can act as a base of acid
35
biochemical reactions take place in aqueous environments
- biological molecules assume their shape and function in response to physical and chemical properties in surrounding water
- water is medium for majority of rxns (an exception is lipid membranes)
- water actively participates in many biochemical rxns bc it can dissociate into H+ and OH-
36
shape of water
- tetrahedral
- free lone pairs of electrons on the water molecule -> these push up the hydrogen atoms
- free lone pairs give partial neg charge to O atom (-.66e)
- hydrogen atoms have partial positive charge (+.33)
- neg on one side and + on one side -> permanent dipole -> allows for hydrogen bonds
37
hydrogen bonds- water
- one water molecule is the hydrogen bond donor and one is the acceptor
- donor- donates a H atom
- acceptor- has free lone pair of electron and accept the H bond
- distance between the H and O atom in the hydrogen bond is 1.77 angstroms (small size of H+ allows it to get very close to O-)
- a single water molecule can donate 2 H (2 H) and accept 2 H (2 lone pair)
- bonds are roughly weak (20kJ/mol) -> when you add them all up its a lot of energy -> gives water its special properties
38
Angstrom
= 1/10 of a nanometer
10 angstroms = 1 nanometer
-ex. .177nm = 1.77 angstrom
39
covalent bond distance between O and H in water
1 angstrom
| .0965 nm
40
hydrogen bond networks in water are constantly switching
- boils at 100C
- not static -> constantly breaking and reforming every 2 x 10^-11 s
- it is able to do this bc of the hydrogen bonds -> gives water its special properties
41
methane
- same tetrahedral shape as water
- similar weight to water
- does not have hydrogen bonds
- boils at -164C (water is 100C)
- there are no interactions between methane so it takes very little amount of energy for methane to go from liquid to gas
42
hydroxyl group
- OH-
- has a free H atom that functions as a donor to the lone pair on water
- also has a lone pair that can accept H bonds
43
carbonyl group
- C=O
- two lone pairs on the O which acts as a H bond acceptor
- can accept two H bonds
- important for secondary structure and peptide bonds
44
carboxylate group
- has 2 O atoms
- can accept 5 H bonds
- functions as a hydrogen bond acceptor
45
ammonium group
- side group of lysine
- N atom with 3 H bonds on it
- 3 H bond donor
46
strength of H bond
- depends on the orientation of the donor and acceptor
- H bond donor is in a linear plane with the acceptor -> strongest form of H bond
- non-linear planar are much weaker bonds
47
hydrophilic
- molecules that tend to dissolve in water
- polar + ionic
- ions surrounded by water molecules are solvated by ordered waters of hydration -> non random orientation -> entropically disfavored
- it dissolves bc the crystal form of NaCl is broken after it is dissolved -> entropically favored
- this breaks ionic bonds and forms H bonds -> favorable
- very exothermic rxn -ΔH -> spontaneous process -> -ΔG
48
hydrophobic
- tend not to dissolve in water
- nonpolar
- molecules tend to aggregate due to hydrophobic effect- tendency of water to minimize its contacts with hydrophobic groups
- dissolving nonpolar substances in nonpolar solvents is entropically driven
49
nonpolar ex
- nonpolar substance (hydrocarbons) dissolved in water (polar)
- transfer them to a nonpolar solvent
- exergonic -ΔG -> spontaneous
- enthalpy is + -> disfavored
- increases entropy when you go from polar to nonpolar solvent
- nonpolar dissolved in nonpolar is entropically driven
50
nonpolar substance dissolved in water- hydrophobic effect
- bc nonpolar substance has no charge there are no favorable interactions
- water tries to minimize contact with nonpolar substance
- forms ordered water caged (clathrates) around nonpolar substance -> aggregates all the nonpolar molecules together and surrounds it
- cage is not favorable bc its organized
- minimizes the SA of the nonpolar substance and maximizes the overall entropy of the water molecules
- more water molecules will be free to form H bonds
- think of the the chicken farmer example (building a fence around a clump of chickens or around each individual chicken)
51
proton hop
- allows water to participate in acid base rxn
- H+ interact with another water molecule and forms H3O+ (hydronium)
- in a chain of water molecules the hydronium ion gives up its extra proton and it "proton hops" along the chain until the water accepts the proton on the other end becoming a hydronium ion
- moves through solution rapidly and constantly
52
dissociation constant of water
Kw = [H+][OH-]
- @ 25C Kw = 10^-14
- concentrations of H+ and OH- are reciprocally related
- ex. if the concentration of a proton is 10^-7 then the OH- concentration is 10^-7
53
pH
=-log[H+]
- low pH -> acidic
- high pH -> basic
- can determine structure
54
acid
substance that can donate a proton
55
base
substance that can accept a proton
56
HA (free acid) + H2O =
H3O+ (conjugate acid) + A- (conjugate base)
57
strength of an acid
- determined by its dissociation constant (Ka)
- dissociation constant are typically written as pK values -> pK=-logK
- ex. Ka = 10^-5 -> pK=5
- dissociation of strong acids shifts rxn to right -> exists as a conjugate base
- weak acids have an equilibrium between the free acid and conjugate base
58
weak acid example
- acetic acid
- monoprotic- donates one H atom
- Ka= 10^-5
- pK=5
- good biological buffer for a lysosome simulation
59
weak acids
- control the pH of a solution using weak acids
- set the pH and control it from moving away from set point
- determined by the relative concentrations by the free acid and conjugate base
- ex. if you want the pH of a solution to be about 5 choose a weak acid with a pK around pH 5 and then calculate the concentration of the free acid and conjugate base we need to add
60
Henderson-hasselbalch equation
pH = pK + log[A-]/[HA]
- used to calculate the pH of weak acids
- calculates the amount of free acid and conjugate base you need to add to reach a certain pH
61
polyprotic
- can donate multiple H atoms
- acids
- monoprotic - 1
- diprotic - 2
- triprotic -3
62
useful weak acids- phosphoric acid
- centered around phosphate
- phosphoric acid has three H atoms
- 3 different pK values for each H atom
- first H atom- pK = 2
- 2nd pK = 7.21 -> biological buffer!
- 3rd pK= 12
- our blood is at a pH of 7.4 so phosphoric acid is a very useful biological buffer for humans
63
biological buffers
- used to help maintain a certain pH
- weak acids with pK close to 7 are useful buffers
- used when you want to mimic the pH inside the cell
- ex. phosphoric acid
64
acetic acid as a buffer
- pK of acetic acid is 4.7
- can function as a buffer for pH between 3.7-5.7
- at this pK value the concentration of free acid and conjugate base are equal
- dissolve free acetic acid in water and add OH- -> conjugate base forms rapidly initially (acetate)
- during this process measure the pH -> pH shoots up rapidly and immediately
- as you add more and more OH- the change in pH slows down and the slope shallows -> buffering region
- at the midpoint the conjugate base and free acid concentration are equal- slope is lowest here
- as we keep adding OH- we quickly drive the free acid all the way to conjugate base
65
buffering compacity
roughly +- 1 of the pK of the weak acid
66
biomolecules can contain multiple ionizable groups
- histidine attached to protein
- 100s of ionizable groups inside a protein
- histidine at pH 5 is protonated
- at pH 7 histidine is deprotonated
- important for the function of enzymes
- as we change pH we change the entire ionization state of the molecule -> affects the shape (H bonds) and its ability to participate in acid base rxn
67
pH optima
- enzymes have a pH optima
- an enzymes pH optima is the pH it functions best at
- due to changes of in the ionization state of the protein and how the H bonds affect 3D shape
68
enzymes
accelerate biochemical rxn
| -speed up
69
what is the OH concentration at room temp when pH = 7
```
pH=-log[H+]
7=-log[H+]
-7 = log [H+]
10^-7 = H+
therefore the OH concentration will also be 10^-7
```
70
1 x 10^-14
= [H+] [OH-] = Kw
71
if the pH = pKa
there will be equal concentrations of the free acid form and the conjugate base
72
amino acids
- chiral -> L and D enantiomers
| - 20 different side chains define 20 different amino acids (19 amino acids and 1 imino acid)
73
structure of amino acid
- central carbon - alpha carbon -> linked to 4 different substituents
1. -amino group- @ physiological pH can accept a proton and become +
2. -carboxylate group- @ physiological pH can lose proton to become -
3. Hydrogen
4. R group- side chain
74
proline
- imino acid
- R group forms a cyclic ring with the alpha carbon and the amino group
- technically a hydrophobic side chain but it has a special structure
- 5 member ring makes it structurally restrictive allowing it to influence structure of proteins
- Pro
- P
75
amino acids are dipolar ions
- amino acids ionize when dissolved in water
- @ physiological pH the amino group is protonated (pH < pKa -> 7 < 9.4)**
- @ physiological pH the carboxylic acid group is deprotonated (pH > pKa -> 7>2.2)**
- zitterions- have + and - charge on a molecule
76
Zwitterions
- molecules bearing both charges (+ -)
- dipolar
- at neutral pH amino acids exist in zwitterion form
- COO-
- NH3+
77
polypeptides
- amino acids are polymerized by a condensation rxn to form polypeptides
- linked by peptide bonds on ribosomes
- linear
- individual amino acids in a polypeptide are called residues
- ALL proteins have a free amino group (N-terminal) and free carboxylate group (C-terminal)
78
if the pH = pKa
there will be equal concentrations of the free acid form and the conjugate base
79
amino acids
- chiral -> L and D enantiomers
| - 20 different side chains define 20 different amino acids
80
structure of amino acid
- central carbon - alpha carbon -> linked to 4 different substituents
1. -amino group- @ physiological pH can accept a proton and become +
2. -carboxylate group- @ physiological pH can lose proton to become -
3. Hydrogen
4. R group- side chain
81
polypeptide
about 10-20 amino acids linked
82
amino acids are dipolar ions
- amino acids ionize when dissolved in water
- @ physiological pH the amino group is protonated (pH < pKa -> 7 < 9.4)
- @ physiological pH the carboxylic acid group is deprotonated (pH > pKa -> 7>2.2)
- zitterions- have + and - charge on a molecule
83
Zwitterions
-molecules bearing both charges (+ -)
84
polypeptides
- amino acids are polymerized by a condensation rxn to form polypeptides
- linked by peptide bonds on ribosomes
- linear
- individual amino acids in a polypeptide are called residues
- all proteins have a free amino group (N-terminal) and free carboxylate group (C-terminal)
85
nonpolar amino acids
- nonionic
- cannot form H bonds
- side chains burry inside the protein bc they are hydrophobic -> aggregate
- do not get protonated and deprotonated
- hydrophobic effect
- alanine
- isoleucine
- phenylalanine
- valine
- leucine
- methionine
- tyrosine
- tryptophan
86
peptide bond
-an amide bond inside a protein
87
oligopeptide
-4 amino acids linked
88
polypeptide
about 10-20 amino acids linked
89
heteropolymers
- 20 amino acids and they can go in any position
| - different combinations
90
uncharged polar side chains
- have hydroxyl, amide, thiol side groups
- glutamine
- theronine
- asparagine
- cystine (special case)
- serine
- hydroxyl- donor
- amide- C=O acts as acceptor; N is a H bond donor -> interacts and H bonds with other proteins
91
glutamine
- C=O & NH2 -amide
- C=O acts as acceptor
- N is a H bond donor to interact with water or other proteins
- uncharged polar side chain
- Glu
- Q
92
serine
- hydroxyl group- donor
- Ser
- S
- uncharged polar side chain
- able to interact with water molecules
93
cysteine
- uncharged polar amino acid
- can form disulfide bonds (covalent bond) with each other
- thiol group (SH)
- d-amino acid
- same as serine except O is switched to S
- this bond loses 2e -> only occurs in an oxidizing environment (majority of inside of cells is reducing so there arnt that many disulfide bonds)
- proteins that are secreted outside the cell are oxidizing and have disulfide bridges -> more stable in our blood
- stabilizes the orientation of 3D structure
- Cys
- C
94
charged polar side chain
- have a charge at normal physiological pH
- hydrophilic
- aspartate
- lysine
- histidine
- glutamate
- arginine
95
aspartate*
- charged polar side chain
- @ physiological pH the aspartic acid will become deprotonated to aspartate -> neg charge (7>3.9)
- O-
- Asp
- D
- acid
96
lysine*
+
- charged polar side chain
- @ physiological pH it become protonated (7<10.5)
- base
- NH3+
- Lys
- K
- pKa= 10.5
97
arginine*
+
- Arg
- R
- charged polar side chain
- pKa- 12.5
- base
98
histidine*
+
- His
- H
- charged polar side chain
- pKa= 6
- base
99
standard nomenclatures
- triple letter code
| - one letter code
100
cysteine
- uncharged polar amino acid
- can form disulfide bonds (covalent bond) with each other
- thiol group (SH)
- this bond loses 2e -> only occurs in an oxidizing environment (majority of inside of cells is reducing so there arnt that many disulfide bonds)
- proteins that are secreted outside the cell are oxidizing and have disulfide bridges -> more stable in our blood
- stabilizes the orientation of 3D structure
- Cys
- C
101
polar side chain
-aspartate
-lysine
-
102
chiral centers
- L and D enantiomers
- compare to glyceraldehyde
- L-glyceraldehyde- OH on the left
- D-glyceraldehyde- OH on the right
- for amino acids instead of OH we judge with NH3
- the top and bottom are going into the screen
- left and right go away from the screen
- steriospecificity
103
lysine*
+
- polar side chain
- @ physiological pH it become protonated (7<10.5)
- NH3+
- Lys
- K
104
D-amino acids
-amino group is on the right away from the screen
105
life is based on chiral molecules
- chirality of molecules can dictate what happens in our cells
- ex. ibuprofen- has 1 chiral center, only one enantiomer is effective at inhibiting pain enzyme -> determines potency
- ex. thalidomide- treats morning sickness, 1 chiral center, caused defects due to one enantiomer
106
standard nomenclatures
- triple letter code
| - one letter code
107
amino group pKa
-@ physiological pH the amino group is protonated (pH < pKa -> 7 < 9.4)**
108
Reversible modifications
- phosphoserine
- phosphothreonine
- phosphotyrosine
- w-N-Methyllarginine
- phosphorylation events- a inorganic phosphate is transferred from ATP to a side chain of an amino acid with a free OH group
- this is done by the enzyme kinases
- enzyme phosphatases or phosphohydrolases can remove the phosphate and reverse the modification
109
biologically active amino acids
- they can be modified to become active
- neurotransmitters or hormones are derived from amino acids
- Tyrosine can be decarboxylated forming the neurotransmitter dopamine which can then be modified into epinephrine
- glutamate can be decarboxylated to form GABA
- histidine can be decarboxylated to form histamine
- tryptophan can be decarboxylated to form serotonin
110
L-amino acids
- only found in nature
- interact with small molecules that are chiral
- amino group on the left away from the screen
- incorporated into proteins
111
alpha amino acids
- are on the alpha carbon
| - alpha carbon is attached to COO- and NH3+
112
glycine
- R group is H
- does not have a chiral center -> no enantiomer
- smallest amino acid
- can fit into either hydrophobic or hydrophilic environments bc its minimally invasive
- Gly
- G
113
non standard amino acids
- amino acid side chains can be modified to make nonstandard amino acids
- enzymes modify amino acid in a protein (posttranslationally) with different chemical groups
- reversible vs. irreversible
114
irreversible modifications
- 4-hydroxy proline
- 5-hydroxylysine
- 6-N-Methyllysine
- 7-Carboxyglutamate
115
Reversible modifications
- phosphoserine
- phosphothreonine
- phosphotyrosine
- w-N-Methyllarginine
- phosphorylation events- a inorganic phosphate is transferred from ATP to a side chain of an amino acid with a free OH group
116
tyrosine
- Tyr
- Y
- ring with OH
- nonpolar
- hydrophobic
- -slightly less hydrophobic due to OH but still hydrophobic bc its big
117
tryptophan
- Trp
- W
- nonpolar
- hydrophobic
- indole ring
- slightly less hydrophobic due to N but still hydrophobic bc its big
118
alpha amino acids
- are on the alpha carbon
| - alpha carbon is attached to COO- and NH3+
119
glycine
- R group is H
| - does not have a chiral center
120
glutamate
- Glu
- E
- charged polar side chain
- neg charge
- pKa= 4.3
- acid
121
lysine
- Lys
- K
- charged polar side chain
- pKa- 10.5
- positive
- base
122
methionine
- Met
- M
- nonpolar
- hydrophobic
- has sulfur group
123
tyrosine
- Tyr
- Y
- ring with OH
- nonpolar
- hydrophobic
124
tryptophan
- Trp
- W
- nonpolar
- hydrophobic
- indole ring
125
theronine
- Thr
- T
- uncharged polar side chain
126
asparagine
- Asn
- N
- uncharged polar side chain
127
glutamate
- Glu
- E
- charged polar side chain
128
lysine
- Lys
- K
- charged polar side chain
129
basic amino acids
- Lysine
- arginine
- histidine
- positive charged side groups
130
acidic amino acids
- aspartate
- glutamate
- negative charged side group
131
why would we want to purify a protein
- if we want to study a particular system we should isolate that system
- surrounding things may influence the data
- ex. if we are counting turkey eggs we dont want other animal eggs there
132
primary structure
- amino acid sequence of that protein
- 100-1000 residues in polypeptides -> must be long enough in order to fold but not too long that it might misfold
- too long polypeptides can be degraded inside the cell
- some large proteins are composed of multiple subunits (multiple polypeptide chains)
- the properties of a protein are affected by the primary structure -> polar, nonpolar residues -> we can take advantage of these properties to isolate
133
purification
- overexpression (significantly increase the amount of protein)- tricks cell to overproduce a specific protein
- isolate this protein by buffering pH range
- lowering the temperature (4C)
- limit exposure to degradative enzymes (proteases) -> do this by lowering temp and adding inhibitors of these enzymes
134
spectroscopy
- measures the concentration of protein by measures the absorption of the protein
- shine light on protein -> measures how much light passes through
- A=log(l0/I)=Ecl
- measures absorbance by measuring path length, concentration, and extinction coefficient
- proteins have bulky side chains that absorb UV light (tryptophan >, tyrosine >, >phenylalanine)
- if the protein has a high number of tryptophan, tyrosine, and phenylalanine the extinction coefficient will be very high and it will absorb a lot of light -> look at primary sequence to tell
135
electrophoresis
- measures the size
- confirms the protein is purified
- SDS PAGE- Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- take acrylamide -> heat -> add a polymerizing agent -> forms a polyacrylamide matrix
- anode and cathode
- place the polyacrylamide matrix in the well and it will moves towards the cathode -> separates protein by size
- SDS- detergent molecule that denatures the protein into a linear chain and coats it with its negative charge
- the smaller proteins are located at the bottom bc they are faster
- larger proteins run slower bc they are interacting with the matrix
- this is not purification
- use markers to determine size or plot the migration of the proteins by the log of their molecular weight NOT size
136
isoelectric point (pI)
- the pH at which the molecule carries no net electric charge
- say we have 10 Asp with a pKa of 3.9 and 20 Lys with a pKa of 10.54 @ pH 7 -> at pH the net charge will be positive -> as we raise the pH there is a point in which some Lys will deprotonate and the overall molecule will carry no net electric charge -> isoelectric point
- isoelectric point is determined experimentally (cant just look at the primary structure bc some side chain are hidden and some residues are closer than others) -> Two dimensional electrophoresis
137
two dimensional electrophoresis
- can resolve complex mixtures
- separating proteins based on pI and molecular weight
- first separate by pI by generating a small polyacrylamide strip with a pH gradient (left pH 9 right pH 3)
- place the strip in an electric field
- place the protein in the gel and see how it migrates
- a positive protein would migrate towards the negative anode (left) -> as it migrates it will approach higher and higher pHs -> as it reaches higher pHs it will deprotonate -> it will continue to migrate until its overall charge becomes neutral -> isoelectric point
- we then place the strip on top of a SDS apparatus -> denatures on proteins -> separate them by size
- good for looking at many proteins all at once
138
chromatography
- how we purify proteins
- involves interaction with mobile and stationary phases
- use selective interaction of a liquid mobile phase with a solid stationary phase
- solid stationary phase that has a matrix that our mobile phase can interact with
- mobile phase is going to be what are molecules are suspended in
- depending on how the molecules interact with the stationary phase they will move either slowly or fast through
- if the molecule reacts strongly with stationary phase- its going to move slowly through the matrix and migrate a little bit
- if the molecule reacts weakly with the stationary phase- its going to move fast through the matrix
- different properties of proteins that different techniques take advantage of: charge, polarity, size, specific binding
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ion exchange chromatography
- separates proteins by charge
- charge is determined by side chains
- stationary phase is either coated with positive or neg charged ions
- anion exchange- neg charged proteins binding to a solid cationic matrix
- cationic exchange- positive charged proteins binding to a solid anionic matrix
- we then judge if they interact weakly or strongly
- we can manipulate the condition to alter the strength of binding of our protein to the matrix by using:
- pH- changes the charge of protein of interest
- salt- compete with our protein for binding
- ex. taking a basic protein with a positive charge -> good for cationic exchange -> we can manipulate by increase the pH and deprotonate the molecules -> reduced charge -> interact more weakly
ex. taking a basic protein with + charge -> cationic exchange -> add a positive salt -> complete for binding and proteins will interact more weakly
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retention time
- the time taken to pass through a chromatography column
| - large positive charged protein will interact strongly with anions and migrate slowly -> longer retention time
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your protein stops moving an isoelectric focusing experiment at pH 8.5. What experimental condition in ion-exchange chromatography would cause your protein to elute from the column with the longest retention time
- a stationary phase crosslinked to a negatively charged group in a buffered solution at pH 4
- you know the protein is positive and basic at neutral pH bc it moved to the left (toward negative) becoming deprotonated and reached its isoelectric point
- if you set the pH to something high like 9 the protein would become negative and no longer interact slowly therefore we must set it to something lower like 4
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hydrophobic interaction chromatography
- separate proteins based on their nonpolarity
- see how proteins interact with a hydrophobic matrix
- hydrophobic proteins will interact strongly
- non hydrophobic proteins will interact weakly
- coat the beads with different hydrophobic chains -> this allows you to manipulate how hydrophobic the matrix is
- longer the chain more hydrophobic (rings are very hydrophobic as well)
- butyl>hexyl>octyl>decyl>phenyl -> increases the strength of the hydrophobic interaction
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gel filtration chromatography
- size exclusion
- separates proteins based on size
- column with funnel on top
- beads are not coated
- beads are porous
- size of protein will determine if it can enter the pores
- proteins that are small enough will enter and interact strongly weaving its way down
- proteins that are too big will not be able to enter and will interact weakly -> will pass through fast
- size exclusion
- small- elute later, longer retention time
- large- elute sooner, shorter retention time
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affinity chromatography
- exploits the specific binding by proteins
- some proteins have very high affinities for certain types of molecules
- most useful technique
- if you have a protein that binds to glucose -> put glucose on the beads bc most other proteins wouldnt bind
- our protein would have very high affinity for bead and interact strongly while the other proteins would elute fast
- wash the protein off by adding a solution with free glucose -> the high amount of free glucose will compete with the glucose on the beads and our protein will elute
- metal affinity chromatography- engineer the protein of interest with a 6 histidine tag
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immobilized metal affinity chromatography
- IMAC
- common purification method
- most powerful purification
- typically the 1st step and paired with another type of purification
- engineer the protein of interest with a 6 histidine tag
- this poly-histidine chain binds tightly to immobilized nickel ions
- our protein of interest will bind to nickel in the column and become immobilized
- take high concentrations of imitizle (similar to histidine tag) -> it will compete for nickel binding and kick off our protein of interest
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SDS-PAGE
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- detergent molecule that denatures the protein into a linear chain and coats it with its negative charge
- analytical procedure to monitor whats happening at each stop of our purification
- electrophoresis separate by size and shape and SDS goes a step further and separates only by molecular weight
- causes protein molecules to lose tertiary structure
- adds negative charge to all polypeptides to induce migration to neg anode
- increases the solubility of non-polar amino acid residues in aqueous solvent
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overview of protein sequence
- you have a protein of unknown sequence (primary structure)
- how many polypeptides are in our protein?
- are the polypeptide sequences linked covalently? -> ex. reduce disulfide bonds ot break them apart
- enzymatically/chemically break polypeptides into fragments
- we want to do this in two separate tubes using two different methods so they fragments are split differently
- then we determine the sequence of the fragments
- overlay the fragments and use computational methods to figure out and recombine the original primary sequence of the protein
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Step 1- protein sequencing: dansyl chloride
- determining the different type of subunits in the protein (how many different polypeptide chains are there)
- use dansyl chloride (bright yellow) that reacts very strongly with primary amines
- there is a primary amine at the N-terminus of every polypeptide chain -> reacts with dansyl chloride
- dansyl polypeptide will turn bright yellow
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Step 2- protein sequence: identifying # of chains and N-terminal residue
- boil the dansyl polypeptide in a strong acid
- acid hydrolyzes all the peptide bonds in our polypeptide sequence
- well have one dansylamino acid (fluorescent) and many other free amino acids
- take the dansylamino acid and run it down a hydrophobic interaction column (dansylamino acid is very hydrophobic)
- watch the bright yellow move down at a specific speed and retention time
- chemically generate all the different dansylated amino acids and run them down a hydrophobic interaction column and record their retention times
- compare these times and identify the amino acid on the N-terminus
- if we see one yellow band we know we have on polypeptide chain -> if there are 2 bands there are two chains...
- this method informs you of the N-terminal residue and the # of peptide chains
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Step 3- protein sequence: breaking disulfide bonds
- if we have multiple polypeptide chains that are linked by disulfide bonds we must cleave them to separate subunits
- Method 1- oxidation rxn using performic acid (rare)
- Method 2- reduce the disulfide bond using dithiothreitol (DTT) or BME (common)
- DTT- has two thiol groups -> reduces the disulfide bond to form free thiol groups of the cysteine residue -> forms its own disulfide bond between two DTT molecules
- to prevent the cysteines from regenerating disulfide bond we treat it with iodoacetate -> carboxymethylation -> irreversible rxn -> cysteines can no longer form disulfide bonds
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Step 4- protein sequence: fragmenting
-now we need to generate fragments enzymatically and chemically
Enzymatically:
-proteases cleave large polypeptides by breaking peptide bonds to produce small fragments
Chemically:
-cyanogen bromide- (toxic) Reacts -> forms a cyclic structure with a peptidyl homoserine lactone group
-when we add water it breaks the peptide bond
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specificity rules of various endopeptidases
-trypsin
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scissle peptide bond
what peptide bond is being cleaved by the protease
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Rn-1 position
- the amino acid to the left of the scissle peptide bond
| - closer to the N-terminus
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Rn position
amino acid to the right of the scissle peptide bond
| -closer to the C-terminus
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trypsin specificity rule
- in the Rn-1 position there is a + residue (Arg or Lys)
- in the Rn position there is any residue other than proline
- highly specific
- there will always be a Arg or Lys at the C-terminus once its cut
- at the N-terminus there can be any amino acid except for proline
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chymotrypsin
- Rn-1 position has a bulky hydrophobic residue (Phe, Trp, Tyr)
- Rn position has anything but proline
- N-terminus has anything but proline
- C-terminus has a hydrophobic bulky residue
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elastase
- Rn-1 position has a small neutral residue (Ala, Gly, Ser, Val)
- Rn position has anything but proline
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Enzymatic cleaving
- proteases cleave large polypeptides by breaking peptide bonds to produce small fragments
- exopeptidases- cleave between the 1st amino acid and the 2nd (cleave off N-terminus)
- endopeptidases- cleave internal bonds (ex. trypsin)
- trypsin- in our intestine -> digests food; cleaves peptide chains with + lysine residue or - arginine residue
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chemical cleaving
- cyanogen bromide- (toxic) Reacts with the side chain methionine -> forms unstable intermediate (+ on S) -> forms a cyclic structure with a peptidyl homoserine lactone group
- when we add water it breaks the peptide bond and we get peptidyl homoserine lactone at the C-terminus
- unknown protein at the N-terminus
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Step 5- protein sequencing: Edman degradation
- sequencing fragments
- removes the first amino acid leaving the rest of the fragment in tact (unlike dansyl chloride)
- uses phenylisothiocyanate (PITC) to react with the primary amine N-terminus @ basic conditions -> forms PTC polypeptide
- PTC is a good leaving group
- use anhydrous trifluoroacetic acid (TFA) (weaker than dansyl chloride acid) -> generates a thiazolinone-amino acid derivative -> retains the shortened polypeptide thats missing the first amino acid
- treat it with aqueous acid -> generates PTH amino acid
- products: PTH-1st amino acid and the rest of the chain
- run this on a hydrophobic interaction column
- separation of PTH-amino acid from rnx mixture using nonpolar solvent extraction
- identify the 1st residue by comparison with standard PTH amino acid chromatography
- repeat this process to identify the 2nd residue so on so forth
- as we keep doing this the peptide gets more and more impure -> we break the peptide to even smaller fragments (at about the 4th residue)
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Step 6- protein sequencing: recombination
-recombine computationally to determine the final polypeptide sequence in our protein
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mass spectrometry
- determines the molecular masses of peptides
- done in the gas phase
- measure the mass/charge ratio of ionized particles in the gas phase
- take our protein molecule and ionize it into the gas phase
- pass it through an electromagnetic field -> scatters -> determine the mass to charge ratio
- determines the mass of the protein at very high precision
- electrospray ionization (ESI) overcomes the propensity of macromolecules to fragment when ionized
- ESI allows us to conduct mass spectrometry for our protein
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tandem mass spectrometry for protein sequencing
- generate fragments of our protein chemically/enzymatically
- determine the mass and identity of those particles using tandem mass spectrometry
- series of two mass spectrometers lined up- ionize proteins into gas phase
- separate ionized peptides using mass spectrometer 1 (MS-1) -> MS-1 determines the molecular weight of the whole fragment
- select one of the fragments (retain the rest in MS-1) and pass it through a collision cell (helium chamber) -> breaks the small polypeptide fragment into further fragments
- determine the size/mass of ionized fragments using MS-2
- we can see the change in weight after the fragmentation
- if the molecular weight lost is = to the weight of a alanine residue then we know that there is alanine present
- accurate and quick
- generation of multiple sets of peptide fragments with overlapping regions
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which is true
- free energy of activation determines the spontaneity of the rxn
- the values of ΔH° and ΔS° can be determined by measuring the equilibrium constant at different initial concentrations of reactants
- enzymes catalyze chemical rxn by lowering gibbs free energy of the products relative to the substrates
- standard conditions require reversible rxn to be at equilibrium
- *the value of ΔG for a reversible chemical rxn changes as the rxn proceeds from the reactants to products
