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1
Q

What drives the protein folding? Why does it do?

A

hydrophobic effect

cause the non-polar groups to aggregate to minimize its contact with water

2
Q

What does the protein folding to the energy and its entropy?

A

from high energy, high entropy to low energy, low entropy

3
Q

What are the other cross-links (bonds) that help stabilize the protein after folding?

A

ion pairs (side chains -R between negative and positive)

disulfide bonds (Cys)

Zn2+

4
Q

Is it true that protein adopt its 3rd comformation before its amino acid fully synthesized?

A

True

5
Q

What can cause to denature protein? How does the factors do?

A

Temperature, pH, detergent, chaotropic agents

disrupt the forces that hold protein in 3rd conformation

6
Q

What happen to the misfolding protein?

A

they can be sent back to get refold or degraded to its components amino acids.

7
Q

What happen to misfolding protein that doesnt degrade or refold? What type of protein does this?

A

they aggregate

Tau and amyloid-beta in Alzheimer’s disease

prion protein in Cow’s disease

alpha-synuclein in Parkinson

8
Q

Why some proteins cant be degraded by protease?

A

because the fibers are resistant to degradation

9
Q

What detergent can denature protein? What does mercaptoethanol do?

A

urea/ salt

cleave sulfide bonds in 3rd conformation.

10
Q

What are 3 types of protein seperation?

A

By charge

By binding specificity

By size

11
Q

Technique for seprate protein by charge

Technique for seprate protein by binding specificity

Technique for seprate protein by size

A

ion exchange chromatography

affinity chromatography

Gel filtration & SDS gel electrophoresis

12
Q

In general, protein seperation requires what for every same technique?

A
Mobile phase (buffered solution with protein in it)
 stationary phase
13
Q

In ion exchange chromatography, what are the stationary phases? What does each do?

A

DEAE ( anion exchange: binding with negative group)

CM (cation exchange: bidning with positive group)

14
Q

In ion exchange chromatography, how do we seperate the protein bound to stationary phase - DEAE?

A

Add high sal-solution or small pH solution

15
Q

In ion exchange chromatography, how do we seperate protein bound with CM phase?

A

high salt-solution and higher pH

16
Q

In what technique separating protein, are the PIs of amino acids important?

A

Ion exchange chromatography

17
Q

What is the most efficient purification method? Why?

A

affinity chromatography

Because the purification depends on the specific binding of the protein and the stationary phase (resin)

18
Q

How do you seperate the protein bound to resin in affinity chromatography?

A

add high salt solution or ligands to compete binding btw protein and resin

19
Q

What properties of proteins do the gel filtration chromatography and SDS-page use to purify proteins?

A

their size and shape

20
Q

Which will move through column faster in gel filtration chromatography: larger or smaller protein?

A

larger proteins

21
Q

What is the only technique that doesnt use high salt/ pH solution to purificate proteins?

A

gel filtration chromatography and SDS-page

22
Q

Process of SDS-page purification

A

SDS detergents make all protein negatively charged

negative charges move toward positive end

bigger proteins move more slowly through the matrix

23
Q

Sequencing steps for a protein

A

1) Purification of protein
2) The individual polypeptide chains are seperated
3) Large polypeptide are broken into smaller pieces (<100 residue)
4) Edman degradation
5) Piece together sequence from overlapping fragments

24
Q

What proteins are used to cleave peptide bonds? What is the characteristic of this protein? What is the process of this called?

A

protease (only cleave at the specific residues)

hydrolysis (adding water)

25
Q

What is mass spectrometry used for?

A

analysis of amino acid sequences

26
Q

Tandem MS-MS. How does this technique determine the amino acid?

A

first MS isolate a fragment, the second determine mass-to-charge ratios of fragment.

By comparing all of fragments, compare those that different by mass of one amino acid to determine sequence.

27
Q

What are techniques to analyze 3rd and 4th protein structure?

A

X-ray crystallography

cryoelectron microscopy

NMR

28
Q

X-ray crystallography

A

Technique to determine the atomic and molecular structure

Crystallized protein is beamed with X-ray, leaving the X-ray diffraction pattern on X-ray film. This will be determined by computer to show 3D map of electron

29
Q

What does the X-ray diffraction pattern show us? What does X-ray crystallography help in protein structure analysis?

A

position and intensities of spots on film

trace the polypeptide backbone and some general side chains

30
Q

cryoelectron microscopy

What is this technique used for?

A

proteins structure are determined by using electron microscopy and then freezed (-196 celcius)
large/complexed proteins (ribosomes)

31
Q

What does NMR determine? What proteins are used?

A

the inter-proton information of protein and its natural conformational flexibility

small protein (<40kDa)

32
Q

Is it true that a polypeptide interact with another protein in chaperone to fold?

A

Yes, its true

33
Q

How many ATP are used in 13s of folding?

A

7 ATP

34
Q

What parts of chaperone help protein to fold? How many polypeptide chains are folded in one chaperone? How many ATPs are used?

A

2 GroEL rings

2

14

35
Q

chaperonin complex consists of

A

2 GroEl/ GroES

36
Q

The chaperonin rxn cycle

A

GroEL binds with 7 ATP and unfolded polypeptide

GroES cap binds, causing the conformational change

cis GroEL ring hydrolyzes 7ATP, release 7 phosphate group

2nd polypeptide and 7ATPs bind to the trans GroEL ring

The cis ring releases 7ADP, GroES caps, folded polypeptide

the trans GroEL ring now can bind GroES cap and repeat step 3-5

37
Q

Where does the unfolded polypeptide bind to GroEL?

A

hydrophobic regions of polypeptide bind to hydrophobic patches of GroEL

38
Q

What happen to GroEL when GroES bind to it?

A

It changes its conformation and release polypeptide into the chamber

39
Q

Why we need the hydrolysis of 7ATP?

A

to cause the missing of gamma-phosphate. This will weakens the interaction btw GreoEL and GreoES

40
Q

What does the 2nd polypeptide and 7ATPs bind to the trans GroEL do?

A

it induces the cis-ring GroEL to release ADP, folded polypeptide, and GroES

41
Q

On average, how many ATPs, time and cycles to fold a protein?

A

24 cycles, 168 ATPs, 91s

42
Q

What is K(d) in ligand-receptor binding? Small K(d)? Large K(d)?

A

dissociation constant

smaller K(d) - the higher infinity of receptor binding to ligands

larger K(d) - the easier (lower affinity) for receptor to dissociate the ligands

43
Q

What is the fraction-bound? What is the characteristic of this fraction?

A

the fraction of number of complex receptor-binding-ligand to the total number of receptors

0<(B)<=1

44
Q

How can we find with K(d)?

A

K(d) is the ligand substrate concentration at which half of receptors saturated.

45
Q

What are the 2 types of receptors for signaling pathways?

A

GPCRs (G protein coupled reactions)

receptor tyrosine kinase

46
Q

What is the signaling pathway of GPCR?

A

Ligand binds to GPCR, causing the conformational change

G protein is activated and trigger enzyme to release second messengers

2nd messengers diffuse to activate or inhibit the activity of targets protein

47
Q

Signaling pathway of receptor tyrosin kinase

A

Ligand bind to the receptor activate its kinase activity

The receptor activate series of kinase-activation

48
Q

Kinase

A

enzyme that transfer phosphoryl group from ATP to another molecue

49
Q

Binding of a subtrate to one site of receptor will affect?

A

the catalytic/ activity of other sites

50
Q

Why we need protein to transport oxygen?

A

oxygen concentration in aqueous solution is low

the oxygen diffusion through thick tissue is inefficient

51
Q

What are the proteins that transport oxygen? The site of each protein present

A

myoglobin (muscle tissue)

hemoglobin (blood)

52
Q

What does myoglobin do? hemoglobin?

A

facilitate oxygen diffusion from capillaries to tissues or used as oxygen storage

exchange O2 and CO2 in lung

53
Q

Is myoglobin required under normal condition? Does aquatic mammals have higher myoglobin concentrations than terrestrials?

A

No, its not required under normal condition

yes (10x higher)

54
Q

Prosthetic group is equivalent to in myoglobin/hemoglobin?

A

heme group

55
Q

How many heme groups does myoglobin have? hemoglobin?

A

one heme group

4 heme groups

56
Q

Myoglobin is a _____ of hemoglobin?

A

a subunit

57
Q

What is the similarity between myoglobin and hemoglobin?

A

heme group

His residue

Val and Phe residue

58
Q

structure of heme group

A

Fe(II) attached to porphyrin ring

59
Q

structure of hemoglobin

A

heme group + protein (4 polypeptides)

60
Q

How does the heme group bind to oxygen? His residue?

A

via the Fe(II)

61
Q

What is the purpose of Phe and Val residue binding to heme group? What happen to these residues after the heme binds to O2?

A

to hold heme group in right position

they move to different position

62
Q

How does the heme group attach to the rest of protein?

A

via covalent/ non-covalnent bonding

63
Q

What does small K(d) mean in oxygen binding?

A

tigher affinity to oxygen. smaller amount of oxygen required to get myoglobin (hemoglobin) saturated

64
Q

What is the fraction O2 binding shape in myoglobin? in hemoglobin?

A

hyperbolic curve

sigmoid curve

65
Q

What state does Hb have w/o oxygen? with oxygen?

A

T-state (dark purple blue)

R-state (red)

66
Q

Which one have smaller K(d) myoglobin or hemoglobin? What does this can tell us?

A

myoglobin has smaller K(d) than hemoglobin (2.6 Torr < 26Torr)

lower affinity at low oxygen concentration

67
Q

What is p(50)

A

the concentration of p(O2) at half-saturated myoglobin/hemoglobin

68
Q

What does the sigmoid curve of hemoglobin indicate? How so?

A

cooperative interactions between different subunits

binding of oxygen to one subunit increase the oxygen affinity to the other subunits

69
Q

At physiological condition, which one is saturated myoglobin or hemoglobin?

A

myoglobin

70
Q

Theoretical maximum cooperativity equal to?

positive cooperativity

negative cooperativity

non-cooperativity

A

of binding sites

n>1 (sigmoid curve) - ligands binding increase affinity of of other binding sites

n<1 - ligands binding decrease affinity

n=1 (hyperbolic curve)

71
Q

Is myoglobin positive, negative or non-cooperative?

A

non-cooperative (n=1)

72
Q

How does the binding of ligand to one site affect to the affinity of other site?

A

due to the conformation changes of one site can influence the conformation changes of other site

73
Q

T-state

R-state

A

the inactive state (no O2 binding) of hemoglobin

the active state (O2 binding)

74
Q

How does the conformation change when heme group bind to oxygen?

A

The binding of oxygen to Fe(II) cause the porphyrin ring to become more flat so that His can come closer to bind with Fe(II)

75
Q

Where does His residue bind to Fe(II)

A

at the nitrogen of histidine

76
Q

If His is replaced by Ala, what will happen?

If His is replaced by Ala and imidazole, what will happen?

A

no cooperativity and poor binding

His -> Gly mutation: no cooperativity but restore binding

77
Q

What curve it will have if hemoglobin has mutation His -> Gly? How do we get this mutation?

A

no sigmoid curve because no cooperativity

add Ala and imidazole

78
Q

What happen when the His binding to Fe(II) undergo conformational change?

A

It also cause the protein conformational changes and trigger the T-state to R-state

79
Q

In T-state, His97 interacts with?

In R-state, His97 interacts with?

A

Thr41

Thr38

80
Q

In T-state, the terminal residues is _____?

in R-state, the terminal residues is _____ and ____ O2?

A

protonated

deprotonated and bind

81
Q

What are the terminal residues that protonated (in T state) and deprotonated (in R state)

A

His 146 (beta 2 chain)

Arg 141 (alpha 1 chain)

82
Q

More Protonation = R-state = _____ pKa and ___ O2 binding?

Deprotonation = T-state = _____ pKa and _____ O2 binding?

A

increase/ less

decrease/ more

83
Q

At ph= 7.6 > ph= 7.4

A

low [H+]

low CO2

K(d) is smaller (higher affinity) and O2 fraction bound is higher

the sigmoid curve shift to left

84
Q

Where does the pH increase in our body?

where does the pH decrease?

A

in lung

in muscle tissues

85
Q

At pH=7.2 < pH=7.4

A

increase [H+]

high CO2

K(d) is larger (lower affinity) and O2 fractiton bound is smaller

O2 affinity decreases to release O2 to the tissue

86
Q

What happen when hemoglobin bind to O2? What happen to the increase [H+] from the muscle tissue?

A

it release [H+] in the blood and become R-state

they bind to Hb to release more oxygen and become T-state

87
Q

How does the BPG change the sigmoid curve of hemoglobin?

A

by binding to cavity of the N-terminus amino acids of beta units leading to the tight T-state

88
Q

What does BPG binding to T-state do?

A

decrease oxygen affinity and favor the unloading of oxygen

89
Q

What is special about hemoglobin in fetal? What does this help the fetal?

A

they have gammar subunit rather than beta subunit and dont have His 143 to bind with BPG.

help fetal hemoglobin binds tighter to oxygen

90
Q

Can the BPG be discarded in normal hemoglobin?

A

Yes, when T-state turns into R-state

91
Q

What happen when we are at high altitude?

A

high BPG to help unload the oxygen to the tissue

the curve shift to the right

92
Q

What is the Bohr effect?

A

the reduction of hemoglobin-oxygen affinity when pH decreases

93
Q

What are the abnormal hemoglobin disease?

A

anemia

bluish skin color

polycythemia

sickle-cell anemia

94
Q

sickle-cell anemia

A

the hemoglobin insoluble filaments distort the blood cell shape, which do not go through capillaries

95
Q

due to what mutation that sickle-cell happen

A

Glu -> Val

96
Q

polycythemia

anemia

A

the increased level of erythrocytes to deliver the same oxygen due to the high affinity to oxygen

the lack of blood cells due to the degradation of unstable hemoglobins

97
Q

bluish skin color

A

the mutation cause the oxidation of Fe(II) to Fe(III) and lead to the decreased cooperativity

98
Q

How does malaria help sickle-cell patient?

A

In malaria patient, low pH cause the excessive deoxygenated Hb, which will be cleared by the spleen

99
Q

How to control the enzyme activity? long term/ medium term and short term?

A

gene regulation (enzyme concentration)

allosteric control (enzyme activity)

activation of zymogens/proenzymes

100
Q

What is the activator/ inhibitor in allosteric regulation?

A

activator: ADP
inhibitor: PEP (feedback inhibition)

101
Q

nucleotide

nucleoside

A

the sugar (deoxyribose/ ribose), phosphate group, the nitrogenous base

sugar and nitrogenous base

102
Q

Where does the nitrogenous base attach sugar?

A

N9 of purine attach to 1’ C of sugar

N1 of pyrimidine attach to 1’C of sugar

103
Q

What are the nitrogenous bases?

A

purine (A,T)

pyrimidine (C,G,U)

104
Q

Where does the phosphate group attach to sugar? Formular for the phosphate group

A

at the 5’C of sugar

HPO4-

105
Q

What is the difference between ribose and deoxyribose?

A

In deoxyribose, H attach to 2’C of sugar

In ribose, only OH attach to 2’C of sugar

106
Q

What is this? How do you know?

A

Ribose

the OH group at 2’C of sugar

107
Q

Where does the Uracil exist? What does this mean?

A

in RNA

A=U instead of A=T in RNA

108
Q

How is uracil and thymine different?

A

by a methyl group (CH3) at C5 position

109
Q

How to name the nucleoside of purine/ pyrimidine?

A

purine = osine (adenosine, guanosine)

pyrimidine = idine (cystidine, thyamidine, uridine)

110
Q

Phosphodiester bond

What does the 5’ end of polypeptide have and 3’ end?

A

the bond of phosphate group between 5’ and 3’ of the sugar

5’ end has phosphate group

3’ end has OH group

111
Q

What form are the bases are found in?

A

keto-form

112
Q

What is the distance between bases stack? in one helical turn (10 pairs)? Width of helix?

A

3.4 Â
34 Â

20 Â

113
Q

What is the width of base pairs (pyrimidine-purine)?

How many H-bonds in A-T, G-C?

A

11Â

2 and 3

114
Q

Where does the H-bond occur in A-T base pair? G-C?

A

1-3 and 6-4

1-3, 2-2, 6-4

115
Q

What is the common form of DNA? What shape is it?

A

beta- DNA

right handed helix

116
Q

When does the B-form DNA turns into A-form DNA? What is the difference btw these 2 forms?

A

when B-form is dehydrated

A-form is wider than B-form

117
Q

What is the characteristics of RNA?

A

Single strand

bases can interact intramolecular to form helix and adopt A-form

118
Q

Genome

chromosome

A

the genetic information of organism

the double stranded genomic DNA

119
Q

When the DNA is denatured, what happen to the absorbance?

What is T(m)?

A

the absorbance increases

the melting temperature of DNA

120
Q

How is the melting curve of duplex DNA affected by Decreasing the ionic strength of the solution?

A

T(m) decreases due to the less shielding of phosphate group

121
Q

How is the melting curve of duplex DNA affected by adding small amount of ethanol?

A

It interacts with hydrophobic regions, causing the unstabilized DNA, T(m) decreases

122
Q

How to increase T(m)

A

increase amount of G-C base pairs

low pH and high salt

123
Q

What significant contribute to the stability of DNA? What other also contribute to the stability?

A

the hydrophobic effect caused by the stacking base pairs

cation shielding (Mg2+, Ca2+, Mn2+, Co2)

124
Q

chromatin

histone

nucleosome

A

DNA/protein complex of chromosomes in eurokarytic cells

the proteins that are used to pack the DNA into nucleosome

histone + DNA

125
Q

Can histon tails be modified? What proteins is it rich in?
What are the histones?

A

Yes,

Lys, Arg, Thr, Ser, His

H1, H2B, H2A, H3 and H4

126
Q

How are the nucleosomes are connected?

A

by H1

127
Q

When is the alpha-helix destabilized? For example?

A

when the amino acids side chain repels each other

polylysine at ph= 7

128
Q

What does the stationary phase in affinity chromatography have?

A

covalent bound protein that protein in mobile phase can bind to

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