Exam 2 Flashcards
(146 cards)
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
What is mass spectrometry used for?
analysis of amino acid sequences
26
Tandem MS-MS. How does this technique determine the amino acid?
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
What are techniques to analyze 3rd and 4th protein structure?
X-ray crystallography
cryoelectron microscopy
NMR
28
X-ray crystallography
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
What does the X-ray diffraction pattern show us? What does X-ray crystallography help in protein structure analysis?
position and intensities of spots on film
trace the polypeptide backbone and some general side chains
30
cryoelectron microscopy
What is this technique used for?
proteins structure are determined by using electron microscopy and then freezed (-196 celcius)
large/complexed proteins (ribosomes)
31
What does NMR determine? What proteins are used?
the inter-proton information of protein and its natural conformational flexibility
small protein (\<40kDa)
32
Is it true that a polypeptide interact with another protein in chaperone to fold?
Yes, its true
33
How many ATP are used in 13s of folding?
7 ATP
34
What parts of chaperone help protein to fold? How many polypeptide chains are folded in one chaperone? How many ATPs are used?
2 GroEL rings
2
14
35
chaperonin complex consists of
2 GroEl/ GroES
36
The chaperonin rxn cycle
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
Where does the unfolded polypeptide bind to GroEL?
hydrophobic regions of polypeptide bind to hydrophobic patches of GroEL
38
What happen to GroEL when GroES bind to it?
It changes its conformation and release polypeptide into the chamber
39
Why we need the hydrolysis of 7ATP?
to cause the missing of gamma-phosphate. This will weakens the interaction btw GreoEL and GreoES
40
What does the 2nd polypeptide and 7ATPs bind to the trans GroEL do?
it induces the cis-ring GroEL to release ADP, folded polypeptide, and GroES
41
On average, how many ATPs, time and cycles to fold a protein?
24 cycles, 168 ATPs, 91s
42
What is K(d) in ligand-receptor binding? Small K(d)? Large K(d)?
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
What is the fraction-bound? What is the characteristic of this fraction?
the fraction of number of complex receptor-binding-ligand to the total number of receptors
0\<(B)\<=1
44
How can we find with K(d)?
K(d) is the ligand substrate concentration at which half of receptors saturated.
45
What are the 2 types of receptors for signaling pathways?
GPCRs (G protein coupled reactions)
receptor tyrosine kinase
46
What is the signaling pathway of GPCR?
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
Signaling pathway of receptor tyrosin kinase
Ligand bind to the receptor activate its kinase activity
The receptor activate series of kinase-activation
48
Kinase
enzyme that transfer phosphoryl group from ATP to another molecue
49
Binding of a subtrate to one site of receptor will affect?
the catalytic/ activity of other sites
50
Why we need protein to transport oxygen?
oxygen concentration in aqueous solution is low
the oxygen diffusion through thick tissue is inefficient
51
What are the proteins that transport oxygen? The site of each protein present
myoglobin (muscle tissue)
hemoglobin (blood)
52
What does myoglobin do? hemoglobin?
facilitate oxygen diffusion from capillaries to tissues or used as oxygen storage
exchange O2 and CO2 in lung
53
Is myoglobin required under normal condition? Does aquatic mammals have higher myoglobin concentrations than terrestrials?
No, its not required under normal condition
yes (10x higher)
54
Prosthetic group is equivalent to in myoglobin/hemoglobin?
heme group
55
How many heme groups does myoglobin have? hemoglobin?
one heme group
4 heme groups
56
Myoglobin is a _____ of hemoglobin?
a subunit
57
What is the similarity between myoglobin and hemoglobin?
heme group
His residue
Val and Phe residue
58
structure of heme group
Fe(II) attached to porphyrin ring
59
structure of hemoglobin
heme group + protein (4 polypeptides)
60
How does the heme group bind to oxygen? His residue?
via the Fe(II)
61
What is the purpose of Phe and Val residue binding to heme group? What happen to these residues after the heme binds to O2?
to hold heme group in right position
they move to different position
62
How does the heme group attach to the rest of protein?
via covalent/ non-covalnent bonding
63
What does small K(d) mean in oxygen binding?
tigher affinity to oxygen. smaller amount of oxygen required to get myoglobin (hemoglobin) saturated
64
What is the fraction O2 binding shape in myoglobin? in hemoglobin?
hyperbolic curve
sigmoid curve
65
What state does Hb have w/o oxygen? with oxygen?
T-state (dark purple blue)
R-state (red)
66
Which one have smaller K(d) myoglobin or hemoglobin? What does this can tell us?
myoglobin has smaller K(d) than hemoglobin (2.6 Torr \< 26Torr)
lower affinity at low oxygen concentration
67
What is p(50)
the concentration of p(O2) at half-saturated myoglobin/hemoglobin
68
What does the sigmoid curve of hemoglobin indicate? How so?
cooperative interactions between different subunits
binding of oxygen to one subunit increase the oxygen affinity to the other subunits
69
At physiological condition, which one is saturated myoglobin or hemoglobin?
myoglobin
70
Theoretical maximum cooperativity equal to?
positive cooperativity
negative cooperativity
non-cooperativity
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
Is myoglobin positive, negative or non-cooperative?
non-cooperative (n=1)
72
How does the binding of ligand to one site affect to the affinity of other site?
due to the conformation changes of one site can influence the conformation changes of other site
73
T-state
R-state
the inactive state (no O2 binding) of hemoglobin
the active state (O2 binding)
74
How does the conformation change when heme group bind to oxygen?
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
Where does His residue bind to Fe(II)
at the nitrogen of histidine
76
If His is replaced by Ala, what will happen?
If His is replaced by Ala and imidazole, what will happen?
no cooperativity and poor binding
His -\> Gly mutation: no cooperativity but restore binding
77
What curve it will have if hemoglobin has mutation His -\> Gly? How do we get this mutation?
no sigmoid curve because no cooperativity
add Ala and imidazole
78
What happen when the His binding to Fe(II) undergo conformational change?
It also cause the protein conformational changes and trigger the T-state to R-state
79
In T-state, His97 interacts with?
In R-state, His97 interacts with?
Thr41
Thr38
80
In T-state, the terminal residues is \_\_\_\_\_?
in R-state, the terminal residues is _____ and ____ O2?
protonated
deprotonated and bind
81
What are the terminal residues that protonated (in T state) and deprotonated (in R state)
His 146 (beta 2 chain)
Arg 141 (alpha 1 chain)
82
More Protonation = R-state = _____ pKa and ___ O2 binding?
Deprotonation = T-state = _____ pKa and _____ O2 binding?
increase/ less
decrease/ more
83
At ph= 7.6 \> ph= 7.4
low [H+]
low CO2
K(d) is smaller (higher affinity) and O2 fraction bound is higher
the sigmoid curve shift to left
84
Where does the pH increase in our body?
where does the pH decrease?
in lung
in muscle tissues
85
At pH=7.2 \< pH=7.4
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
What happen when hemoglobin bind to O2? What happen to the increase [H+] from the muscle tissue?
it release [H+] in the blood and become R-state
they bind to Hb to release more oxygen and become T-state
87
How does the BPG change the sigmoid curve of hemoglobin?
by binding to cavity of the N-terminus amino acids of beta units leading to the tight T-state
88
What does BPG binding to T-state do?
decrease oxygen affinity and favor the unloading of oxygen
89
What is special about hemoglobin in fetal? What does this help the fetal?
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
Can the BPG be discarded in normal hemoglobin?
Yes, when T-state turns into R-state
91
What happen when we are at high altitude?
high BPG to help unload the oxygen to the tissue
the curve shift to the right
92
What is the Bohr effect?
the reduction of hemoglobin-oxygen affinity when pH decreases
93
What are the abnormal hemoglobin disease?
anemia
bluish skin color
polycythemia
sickle-cell anemia
94
sickle-cell anemia
the hemoglobin insoluble filaments distort the blood cell shape, which do not go through capillaries
95
due to what mutation that sickle-cell happen
Glu -\> Val
96
polycythemia
anemia
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
bluish skin color
the mutation cause the oxidation of Fe(II) to Fe(III) and lead to the decreased cooperativity
98
How does malaria help sickle-cell patient?
In malaria patient, low pH cause the excessive deoxygenated Hb, which will be cleared by the spleen
99
How to control the enzyme activity? long term/ medium term and short term?
gene regulation (enzyme concentration)
allosteric control (enzyme activity)
activation of zymogens/proenzymes
100
What is the activator/ inhibitor in allosteric regulation?
activator: ADP
inhibitor: PEP (feedback inhibition)
101
nucleotide
nucleoside
the sugar (deoxyribose/ ribose), phosphate group, the nitrogenous base
sugar and nitrogenous base
102
Where does the nitrogenous base attach sugar?
N9 of purine attach to 1' C of sugar
N1 of pyrimidine attach to 1'C of sugar
103
What are the nitrogenous bases?
purine (A,T)
pyrimidine (C,G,U)
104
Where does the phosphate group attach to sugar? Formular for the phosphate group
at the 5'C of sugar
HPO4-
105
What is the difference between ribose and deoxyribose?
In deoxyribose, H attach to 2'C of sugar
In ribose, only OH attach to 2'C of sugar
106
What is this? How do you know?
Ribose
the OH group at 2'C of sugar
107
Where does the Uracil exist? What does this mean?
in RNA
A=U instead of A=T in RNA
108
How is uracil and thymine different?
by a methyl group (CH3) at C5 position
109
How to name the nucleoside of purine/ pyrimidine?
purine = osine (adenosine, guanosine)
pyrimidine = idine (cystidine, thyamidine, uridine)
110
Phosphodiester bond
What does the 5' end of polypeptide have and 3' end?
the bond of phosphate group between 5' and 3' of the sugar
5' end has phosphate group
3' end has OH group
111
What form are the bases are found in?
keto-form
112
What is the distance between bases stack? in one helical turn (10 pairs)? Width of helix?
3.4 Â
34 Â
20 Â
113
What is the width of base pairs (pyrimidine-purine)?
How many H-bonds in A-T, G-C?
11Â
2 and 3
114
Where does the H-bond occur in A-T base pair? G-C?
1-3 and 6-4
1-3, 2-2, 6-4
115
What is the common form of DNA? What shape is it?
beta- DNA
right handed helix
116
When does the B-form DNA turns into A-form DNA? What is the difference btw these 2 forms?
when B-form is dehydrated
A-form is wider than B-form
117
What is the characteristics of RNA?
Single strand
bases can interact intramolecular to form helix and adopt A-form
118
Genome
chromosome
the genetic information of organism
the double stranded genomic DNA
119
When the DNA is denatured, what happen to the absorbance?
What is T(m)?
the absorbance increases
the melting temperature of DNA
120
How is the melting curve of duplex DNA affected by Decreasing the ionic strength of the solution?
T(m) decreases due to the less shielding of phosphate group
121
How is the melting curve of duplex DNA affected by adding small amount of ethanol?
It interacts with hydrophobic regions, causing the unstabilized DNA, T(m) decreases
122
How to increase T(m)
increase amount of G-C base pairs
low pH and high salt
123
What significant contribute to the stability of DNA? What other also contribute to the stability?
the hydrophobic effect caused by the stacking base pairs
cation shielding (Mg2+, Ca2+, Mn2+, Co2)
124
chromatin
histone
nucleosome
DNA/protein complex of chromosomes in eurokarytic cells
the proteins that are used to pack the DNA into nucleosome
histone + DNA
125
Can histon tails be modified? What proteins is it rich in?
What are the histones?
Yes,
Lys, Arg, Thr, Ser, His
H1, H2B, H2A, H3 and H4
126
How are the nucleosomes are connected?
by H1
127
When is the alpha-helix destabilized? For example?
when the amino acids side chain repels each other
polylysine at ph= 7
128
What does the stationary phase in affinity chromatography have?
covalent bound protein that protein in mobile phase can bind to
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