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Biochemistry > Biochem 1 > Flashcards

Biochem 1 Flashcards

1
Q

biochemistry

A

-study of life at the molecular level

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2
Q

thermodynamics

A

-the relationship between energy, work, and heat

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3
Q

energy

A

capacity to do work

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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

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5
Q

heat

A

transfer of energy as a result of a difference in temperature
-disorganized- difference in temp

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6
Q

system

A
  • what we define

- what were studying

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7
Q

surroundings

A

-everything else in the universe other than the system

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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
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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
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10
Q

enthalpy

A
  • thermodynamic potential of a system
  • H
  • H= U + PV
  • at constant pressure enthalpy equals heat
  • defined in kJ
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11
Q

exothermic

A
  • release of energy
  • change of enthalpy is negative
  • -ΔH
  • release heat
  • ex. -10kJ
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12
Q

endothermic

A
  • requires addition of heat
  • change of enthalpy is positive
  • +ΔH
  • ex. 10kJ
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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
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14
Q

nonspontaneous process

A
  • requires energy for process to occur

- ΔH > TΔS

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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
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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

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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
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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
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19
Q

exergonic

A

-if change in G is less than or equal to 0 the process will occur spontaneously

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20
Q

endergonic

A

-if change in G is greater than 0 the process will not occur spontaneously

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21
Q

negative H, positive S

A
  • enthalpically favored and entropically favored
  • spontaneous at all temperatures
  • exothermic
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22
Q

negative H, negative S

A
  • enthalpically favored and entropically unfavored
  • spontaneous at temperatures below T= Δ H / Δ S
  • exothermic
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23
Q

positive H, positive S

A
  • enthalpically unfavored and entropically favored
  • spontaneous at temperatures above T= ΔH/ΔS
  • endothermic
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24
Q

positive H, negative S

A
  • enthalpically unfavored and entropically unfavored
  • nonspontaneous at all temperatures
  • endothermic
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25
Q

where does energy come from- ATP

A
  • 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
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26
Q

concentrations of the reactions and products

A
  • 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
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27
Q

standard free energy change

A
  • constant

- ΔG^degree

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28
Q

standard condition

A
  • 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
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29
Q

equilibrium

A
  • 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
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30
Q

Van’t Hoff equation

A
  • 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
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31
Q

positive y-intercept, positive slope

A
  • positive slope means that ΔH is negative
  • if ΔH is negative -> exothermic
  • slope = -ΔH/R
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32
Q

hydrophobic effect

A
  • 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
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33
Q

intracellular process

A
  • 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
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34
Q

water

A
  • 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
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35
Q

biochemical reactions take place in aqueous environments

A
  • 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-
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36
Q

shape of water

A
  • 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
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37
Q

hydrogen bonds- water

A
  • 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
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38
Q

Angstrom

A

= 1/10 of a nanometer
10 angstroms = 1 nanometer
-ex. .177nm = 1.77 angstrom

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39
Q

covalent bond distance between O and H in water

A

1 angstrom

.0965 nm

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40
Q

hydrogen bond networks in water are constantly switching

A
  • 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
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41
Q

methane

A
  • 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
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42
Q

hydroxyl group

A
  • 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
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43
Q

carbonyl group

A
  • 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
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44
Q

carboxylate group

A
  • has 2 O atoms
  • can accept 5 H bonds
  • functions as a hydrogen bond acceptor
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45
Q

ammonium group

A
  • side group of lysine
  • N atom with 3 H bonds on it
  • 3 H bond donor
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46
Q

strength of H bond

A
  • 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
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47
Q

hydrophilic

A
  • 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
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48
Q

hydrophobic

A
  • 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
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49
Q

nonpolar ex

A
  • 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
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50
Q

nonpolar substance dissolved in water- hydrophobic effect

A
  • 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)
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51
Q

proton hop

A
  • 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
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52
Q

dissociation constant of water

A

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
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53
Q

pH

A

=-log[H+]

  • low pH -> acidic
  • high pH -> basic
  • can determine structure
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54
Q

acid

A

substance that can donate a proton

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55
Q

base

A

substance that can accept a proton

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56
Q

HA (free acid) + H2O =

A

H3O+ (conjugate acid) + A- (conjugate base)

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57
Q

strength of an acid

A
  • 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
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58
Q

weak acid example

A
  • acetic acid
  • monoprotic- donates one H atom
  • Ka= 10^-5
  • pK=5
  • good biological buffer for a lysosome simulation
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59
Q

weak acids

A
  • 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
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60
Q

Henderson-hasselbalch equation

A

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
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61
Q

polyprotic

A
  • can donate multiple H atoms
  • acids
  • monoprotic - 1
  • diprotic - 2
  • triprotic -3
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62
Q

useful weak acids- phosphoric acid

A
  • 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
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63
Q

biological buffers

A
  • 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
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64
Q

acetic acid as a buffer

A
  • 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
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65
Q

buffering compacity

A

roughly +- 1 of the pK of the weak acid

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66
Q

biomolecules can contain multiple ionizable groups

A
  • 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
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67
Q

pH optima

A
  • 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
Q

enzymes

A

accelerate biochemical rxn

-speed up

69
Q

what is the OH concentration at room temp when pH = 7

A 
pH=-log[H+]
7=-log[H+]
-7 = log [H+]
10^-7 = H+
therefore the OH concentration will also be 10^-7
70
Q

1 x 10^-14

A

= [H+] [OH-] = Kw

71
Q

if the pH = pKa

A

there will be equal concentrations of the free acid form and the conjugate base

72
Q

amino acids

A
  • chiral -> L and D enantiomers

- 20 different side chains define 20 different amino acids (19 amino acids and 1 imino acid)

73
Q

structure of amino acid

A
  • 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
Q

proline

A
  • 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
Q

amino acids are dipolar ions

A
  • 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
Q

Zwitterions

A
  • molecules bearing both charges (+ -)
  • dipolar
  • at neutral pH amino acids exist in zwitterion form
  • COO-
  • NH3+
77
Q

polypeptides

A
  • 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
Q

if the pH = pKa

A

there will be equal concentrations of the free acid form and the conjugate base

79
Q

amino acids

A
  • chiral -> L and D enantiomers

- 20 different side chains define 20 different amino acids

80
Q

structure of amino acid

A
  • 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
Q

polypeptide

A

about 10-20 amino acids linked

82
Q

amino acids are dipolar ions

A
  • 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
Q

Zwitterions

A

-molecules bearing both charges (+ -)

84
Q

polypeptides

A
  • 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
Q

nonpolar amino acids

A
  • 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
Q

peptide bond

A

-an amide bond inside a protein

87
Q

oligopeptide

A

-4 amino acids linked

88
Q

polypeptide

A

about 10-20 amino acids linked

89
Q

heteropolymers

A
  • 20 amino acids and they can go in any position

- different combinations

90
Q

uncharged polar side chains

A
  • 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
Q

glutamine

A
  • 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
Q

serine

A
  • hydroxyl group- donor
  • Ser
  • S
  • uncharged polar side chain
  • able to interact with water molecules
93
Q

cysteine

A
  • 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
Q

charged polar side chain

A
  • have a charge at normal physiological pH
  • hydrophilic
  • aspartate
  • lysine
  • histidine
  • glutamate
  • arginine
95
Q

aspartate*

A
  • charged polar side chain
  • @ physiological pH the aspartic acid will become deprotonated to aspartate -> neg charge (7>3.9)
  • O-
  • Asp
  • D
  • acid
96
Q

lysine*

A

+

  • charged polar side chain
  • @ physiological pH it become protonated (7<10.5)
  • base
  • NH3+
  • Lys
  • K
  • pKa= 10.5
97
Q

arginine*

A

+

  • Arg
  • R
  • charged polar side chain
  • pKa- 12.5
  • base
98
Q

histidine*

A

+

  • His
  • H
  • charged polar side chain
  • pKa= 6
  • base
99
Q

standard nomenclatures

A
  • triple letter code

- one letter code

100
Q

cysteine

A
  • 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
Q

polar side chain

A

-aspartate
-lysine
-

102
Q

chiral centers

A
  • 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
Q

lysine*

A

+

  • polar side chain
  • @ physiological pH it become protonated (7<10.5)
  • NH3+
  • Lys
  • K
104
Q

D-amino acids

A

-amino group is on the right away from the screen

105
Q

life is based on chiral molecules

A
  • 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
Q

standard nomenclatures

A
  • triple letter code

- one letter code

107
Q

amino group pKa

A

-@ physiological pH the amino group is protonated (pH < pKa -> 7 < 9.4)**

108
Q

Reversible modifications

A
  • 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
Q

biologically active amino acids

A
  • 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
Q

L-amino acids

A
  • only found in nature
  • interact with small molecules that are chiral
  • amino group on the left away from the screen
  • incorporated into proteins
111
Q

alpha amino acids

A
  • are on the alpha carbon

- alpha carbon is attached to COO- and NH3+

112
Q

glycine

A
  • 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
Q

non standard amino acids

A
  • 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
Q

irreversible modifications

A
  • 4-hydroxy proline
  • 5-hydroxylysine
  • 6-N-Methyllysine
  • 7-Carboxyglutamate
115
Q

Reversible modifications

A
  • 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
Q

tyrosine

A
  • Tyr
  • Y
  • ring with OH
  • nonpolar
  • hydrophobic
  • -slightly less hydrophobic due to OH but still hydrophobic bc its big
117
Q

tryptophan

A
  • Trp
  • W
  • nonpolar
  • hydrophobic
  • indole ring
  • slightly less hydrophobic due to N but still hydrophobic bc its big
118
Q

alpha amino acids

A
  • are on the alpha carbon

- alpha carbon is attached to COO- and NH3+

119
Q

glycine

A
  • R group is H

- does not have a chiral center

120
Q

glutamate

A
  • Glu
  • E
  • charged polar side chain
  • neg charge
  • pKa= 4.3
  • acid
121
Q

lysine

A
  • Lys
  • K
  • charged polar side chain
  • pKa- 10.5
  • positive
  • base
122
Q

methionine

A
  • Met
  • M
  • nonpolar
  • hydrophobic
  • has sulfur group
123
Q

tyrosine

A
  • Tyr
  • Y
  • ring with OH
  • nonpolar
  • hydrophobic
124
Q

tryptophan

A
  • Trp
  • W
  • nonpolar
  • hydrophobic
  • indole ring
125
Q

theronine

A
  • Thr
  • T
  • uncharged polar side chain
126
Q

asparagine

A
  • Asn
  • N
  • uncharged polar side chain
127
Q

glutamate

A
  • Glu
  • E
  • charged polar side chain
128
Q

lysine

A
  • Lys
  • K
  • charged polar side chain
129
Q

basic amino acids

A
  • Lysine
  • arginine
  • histidine
  • positive charged side groups
130
Q

acidic amino acids

A
  • aspartate
  • glutamate
  • negative charged side group
131
Q

why would we want to purify a protein

A
  • 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
Q

primary structure

A
  • 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
Q

purification

A
  • 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
Q

spectroscopy

A
  • 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
Q

electrophoresis

A
  • 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
Q

isoelectric point (pI)

A
  • 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
Q

two dimensional electrophoresis

A
  • 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
Q

chromatography

A
  • 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
139
Q

ion exchange chromatography

A
  • 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
140
Q

retention time

A
  • the time taken to pass through a chromatography column

- large positive charged protein will interact strongly with anions and migrate slowly -> longer retention time

141
Q

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
  • 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
142
Q

hydrophobic interaction chromatography

A
  • 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
143
Q

gel filtration chromatography

A
  • 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
144
Q

affinity chromatography

A
  • 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
145
Q

immobilized metal affinity chromatography

A
  • 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
146
Q

SDS-PAGE

A
  • 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
147
Q

overview of protein sequence

A
  • 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
148
Q

Step 1- protein sequencing: dansyl chloride

A
  • 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
149
Q

Step 2- protein sequence: identifying # of chains and N-terminal residue

A
  • 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
150
Q

Step 3- protein sequence: breaking disulfide bonds

A
  • 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
151
Q

Step 4- protein sequence: fragmenting

A

-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

152
Q

specificity rules of various endopeptidases

A

-trypsin

153
Q

scissle peptide bond

A

what peptide bond is being cleaved by the protease

154
Q

Rn-1 position

A
  • the amino acid to the left of the scissle peptide bond

- closer to the N-terminus

155
Q

Rn position

A

amino acid to the right of the scissle peptide bond

-closer to the C-terminus

156
Q

trypsin specificity rule

A
  • 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
157
Q

chymotrypsin

A
  • 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
158
Q

elastase

A
  • Rn-1 position has a small neutral residue (Ala, Gly, Ser, Val)
  • Rn position has anything but proline
159
Q

Enzymatic cleaving

A
  • 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
160
Q

chemical cleaving

A
  • 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
161
Q

Step 5- protein sequencing: Edman degradation

A
  • 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)
162
Q

Step 6- protein sequencing: recombination

A

-recombine computationally to determine the final polypeptide sequence in our protein

163
Q

mass spectrometry

A
  • 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
164
Q

tandem mass spectrometry for protein sequencing

A
  • 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
165
Q

which is true

A
  • 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

Biochemistry (8 decks)

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