1
Q
Three ways to determine protein structure
A
- Protein crystallography
- Cyro-electron microscopy
- NMR spectroscopy
2
Q
Non-chiral amino acid
A
Glycine
3
Q
Amino acids that can absorb UV light
A
Tryptophan
Tyrosine
Phenylalanine
4
Q
Negatively charged side chains are acidic/basic
A
Acidic
5
Q
Pka = pH when group is ___% ionised
A
50%
6
Q
pH is pI when net charge is
A
0
7
Q
RNA → protein occurs at
A
Ribosome
8
Q
Phosphatases function
A
Removes phosphate via hydrolysis
9
Q
Hydroxylation often has ______ involved
A
Proline
Lysine
10
Q
Carboxylation often has _______ involved
A
Glutamate
11
Q
Glycosylation often has _______ involved
A
Threonine
Asparagine
12
Q
Peptide bond has ___% double bond character
A
40%
13
Q
Peptide bond has a rotational barrier of ___ kJ/mol
A
80 kJ/mol
14
Q
Rotational barrier
A
Energy difference between the most and least stable conformations when a chemical rotates around a bond
15
Q
Polypeptide = ___ amino acids
A
> 50 amino acids
16
Q
ɸ
A
Phi
17
Q
ψ
A
Psi
18
Q
Bond angle between N and Cₐ is called
A
Phi
19
Q
Bond angle between Cₐ and C’ is called
A
Psi
20
Q
Psi and phi bond angles can be between
A
-180° and +180°
21
Q
Chain angle between C’ and N is called
A
ω (omega)
22
Q
Alpha helix is left/right handed
A
Right
23
Q
Phi rotations lead to ________ collisions
A
O-O
24
Q
Psi rotations lead to ________ collisions
A
NH-NH
25
Peptide bond is _______ Å shorter/longer than Cₐ- N
0.13
Shorter
26
Trans peptide bond angle
~180°
27
Cis peptide bond angle
~0°
28
Alpha helix hydrogen bond length between O-N
~2.9 Å
29
Alpha helices have ____ residues per turn
3.6
30
Hydrogen bonds have energy of
12-18 kJ/mol
31
Alpha helices have rise of ______ per turn
5.4 Å
32
d refers to
Distance between n and n+4
33
d = ____ per residue
1.5 Å
34
In alpha helices psi =
~ -47°
35
Helix breaker examples
Glycine
Proline
36
In alpha helices, each amino acid side chain is separated by ____°
100°
37
______ strands per sheet
2-10
38
Average strand length
6 amino acids
39
Maximum stand length
15 amino acids
40
Turns have ___ residues
3-4
41
Almost ___% of protein residues are in turns
30%
42
Type 1 turns
Hydrogen bond is between residues n and n+3
43
Type 2 turns
Hydrogen bond is between n and n+2
44
Most common type of turn
Type 1
45
Supersecondary structure
Elements of secondary structures that are connected by turns, loops or coils
46
Helix-turn-helix commonly found in
DNA binding proteins
47
Helix-loop-helix is a
Calcium binding protein
48
Beta-hairpins
Two antiparallel beta strands next to each other
49
Amphipathic
Has both hydrophilic and hydrophobic parts
50
Alpha-domain family
Amphipathic helices with side chains in hydrophobic core
51
Most common in alpha-helices family
Four helix bundle
52
Helices like to sit ____° away from each other
20-50°
53
Alpha/beta family example
Alpha/beta horseshoe fold
54
Chaperone independent
Polypeptide is made and folds up
55
Chaperone independent and chaperone dependent make up ___% of protein folding
85%
56
Chaperonin dependent makes up ___% of protein folding
15%
57
Prions (PrP)
Proteins that cause misfolding of normal versions of the protein
58
Oxidoreductases
Redox enzymes
59
Transferases
Enzymes that transfer functional groups
60
Hydrolases
Enzymes that do hydrolysis
61
Lyases
Enzymes that do non-hydrolic breaking/making of bonds
62
Isomerases
Enzymes that transfer substrates to an isomeric form
63
Ligases
Enzymes that join two molecules together, usually coupled to ATP cleavage
64
Two classes of cofactor
Metal ions
Coenzymes
65
Activation energy is lowered by
Destabilising ground state
Stabilising transition state
66
Enzyme/chemistry specific catalytic mechanisms
Acid-base
Metal ion
Covalent
67
First linear part of graph is ____ order kinetics do rate does/doesn't depend on [S]
First order
Does
68
Second part of graph is _____ order kinetics, so rate does/doesn't depend on [S]
Zero order
Doesn't
69
Vmax
Maximum velocity when [S] = ∞
70
Km
[S] needed to reach half Vmax
71
Haldane's steady state assumption
The rate of ES formation = rate of its breakdown
72
E + S ⇌ ES
Slow or fast?
Fast
73
ES → E + P
Fast or slow?
Slow
74
Michaelis constant
Km
75
Low Km means
High affinity between E and S
76
ES dissociation constant
Km = K₋₁/K₁
77
In Lineweaver-Burk plot, y intercept =
1/Vmax
78
In Lineweaver-Burk plot, x intercept =
-1/Km
79
Good enzyme has ___ kcat and ____ Km
High kcat
Low Km
80
kcat
Number of substrate molecules converted to product per unit time
81
kcat =
= k₂
= Vmax/[E]ₜ
82
Overall measure of enzyme efficiency
kcat/Km
83
Diffusion controlled limit
When reaction is so fast the rate is determined by the rate at which E and S diffuse together
84
Absolute upper limit for enzymes
~ 10⁹ s⁻¹ M⁻¹
85
Enzymes with kcat/Km above ____ are perfect
10⁸ s⁻¹ M⁻¹
86
Consequence of competitive inhibition
Slows reaction down
87
In competitive, Vmax decreases/stays the same and Km increases/stays the same
Vmax stays the same
Km increases
88
In non-competitive, Vmax decreases/stays the same, and Km increases/stays the same
Vmax decreases
Km stays the same
89
In mixed inhibitors, Vmax decreases/stays the same and Km changes/stays the same
Vmax decreases
Km changes
90
Proteolytic cleavage
Made in longer form, cleaved when used
91
Solubility of oxygen in saline
0.2 mmol L⁻¹
92
Oxygen is stored in muscle in
Myoglobin
93
Oxygen in muscle is gone in
7 seconds
94
Solubility of oxygen in muscle
0.5 - 0.7 mmol L⁻¹
95
Myoglobin primary structure
~150 amino acids
96
Myoglobin secondary structure
8 alpha helices (labelled A-H) connected by loops
97
Which amino acid does heme interact with in myoglobin?
Histidine F8
98
In myoglobin, the six Fe coordinate bonds go to
4 - N atoms of heme
1 - N of histidine F8
1 - oxygen
99
What gives myoglobin its red colour?
Molecular electronic orbitals of the heme
100
Oxygenated/deoxygenated myoglobin gives two peaks on a spectroscopy graph (as opposed to one)
Oxygenated
101
Oxygenated/deoxygenated myoglobin is bright red
Oxygenated
102
Quaternary structure of hemoglobin
Non-covalent tetramer
103
Does myoglobin or hemoglobin bind oxygen more weakly?
Hemoglobin
104
Partial pressure of oxygen in lungs
~100 Torr
105
Partial pressure of oxygen in resting muscle
~20 Torr
106
Shape of myoglobin oxygen saturation curve
Hyperbolic
107
Shape of hemoglobin oxygen saturation curve
Sigmoidal
108
Which becomes saturated with oxygen at high concs?
Hemoglobin
109
T state = _____ affinity for oxygen
Low
110
R state = _______ affinity for oxygen
High
111
Which state is when oxygen is bound?
R
112
Hemoglobin is predominantly in ____ state
R state
113
Where does BPG bind to hemoglobin?
Allosteric site between beta chains
114
Lower pH favours ___ state
T state
115
How does CO₂ stabilise T state?
Lowering pH
Binds to amino terminus
116
When is beta hemoglobin expressed?
After birth
117
Foetal hemoglobin holds oxygen weaker/tighter
Tighter
118
Foetal hemoglobin is less/more sensitive to BPG
Less
119
HbS
Abnormal hemoglobin that causes sickle cell anemia
120
HbS variant
E6V
In beta chain
121
Warfarin low doses effect
Prevents blood clots post heart attack or stroke
122
Which has only one active site, receptors or enzymes?
Enzymes
123
Endogenous ligands are usually
Antagonists
124
Synaptic
Really close
125
Paracrine
Acts of nearby cells
126
Endocrine
Released in one space, picked up in another
127
Phosphodiesterases
Break down cAMP and cGMP
128
Gɑₛ
Stimulatory G protein
129
What do Gɑₛ and Gɑᵢ effect?
Adenylate cyclase
130
Adenylate cyclase function
Makes cAMP
131
When GDP is bound to G protein, it is
Inactive
132
When GTP is bound to G protein, it is
Active
133
GTP replaces GDP when
G protein interacts with receptor
134
What hydrolyses GTP to GDP?
GTPase
135
How do RTKs start signal transduction?
Phosphorylation of adaptor protein
136
Transcription
DNA → RNA
137
Translation
RNA → Protein
138
Transcribed region
Part of gene that is copied into mRNA
139
Where are introns and exons found?
Transcribed region
140
RNA polymerase function
Makes mRNA
141
DNA has ___ base where RNA has ___ base
DNA = T
RNA = U
142
Where is mRNA made?
Nucleus
143
Where is mRNA translated?
Cytoplasm
144
Function of promoter region
Controls gene transcription
145
Promoter region contains
Transcription factor binding sites
146
What are binding sites in promoter region?
Short specific DNA sequences
147
Transcription factor function
Binds to promoter and flags down RNA polymerase
148
Two kinds of transcription factors
Activators
Repressors
149
How many cells in the human body?
~ 30 trillion
150
How many different types of cell in the human body?
~200
151
How many protein coding genes?
~21,000
152
How many proteins can be made?
~85,000
153
Alternative splicing
Exons spliced in different combinations
154
How many genes does each cell type express?
~11,000 - 17,000
155
How many genes are expressed in all cells?
~10,000
156
How many genes are expressed only in specific cells?
~1,000 - 2,000
157
Ribosomes made of
rRNA and proteins
158
What occurs in ribosomes?
Translation
159
C always paired with
G
160
tRNA function
Reads codons in mRNA
161
tRNA structure
Anti-codon on one end, amino acid on the other
162
Three stages of translation
1. Initiation
2. Elongation
3. Termination
163
Initiation
Ribosome, mRNA and first tRNA come together to form translation initiation complex
164
First tRNA always codes for
Methionine
165
Elongation
Ribosomes move along mRNA adding amino acids
166
Termination
Stop codon causes translation complex to break apart
167
How many possible codons?
4³ = 64
168
Which amino acids can't be coded for by multiple codons?
Methionine
Tryptophan
169
Codon redundancy
Most amino acids are specified by more than one codon
170
Humans are ___% different from one another
0.1%
171
How many base pairs is the whole (diploid)human genome made up of?
6 billion
172
Alleles
Different forms of a variant in the same location
173
SNP
Single base change in the DNA sequence
174
Where are coding variants located?
Exon
175
List non-coding variants
Intergenic variant
Regulatory variant
Intron variants
176
Where are intergenic variants located?
Stretch of DNA between two genes
177
Where are regulatory variants located?
Promoter region
178
Non-coding variants can change
Expression of gene
179
Coding variants can change
Amino acid sequence and potentially function of the protein
180
Synonymous
Altered codon specifies for the same amino acid
181
Missense
Altered codon specifies for one different amino acid
182
Nonsense
Altered codon specifies early stop
183
Diploid
Cells contain two complete sets of chromosomes
184
Homologous pair
Two of each chromosome that diploid organisms have
185
Phenotype
Observable trait
186
Genotype
Combination of alleles a person has for a particular genetic variant
187
Homozygote dominant
Double dominant
188
Heterozygote dominant
One of each
189
Gain of function allele often shown in which inheritance pattern?
Dominant
190
Genotyping
Determining the specific genetic alleles in an organism's DNA
191
PCR primer
Small sequence of DNA complementary to specific sequence surrounding DNA of interest
192
What does PCR-RFLP use to determine genotype?
PCR
Restriction enzymes
Gel electrophoresis
193
Restriction enzymes
Proteins isolated from bacteria that cut DNA
194
Monogenic diseases affect ___% of people
6%
195
Proto-oncogenes
Normal cells that regulate cell growth and division
196
Oncogene mutation is ______-of-function
Gain
197
___% of cancers caused by inherited genetic variants
10%
198
Recombinant DNA technologies
Joining bits of DNA
199
Reporter gene function
Produces a signal to monitor gene expression
200
Plasmid
Circular piece of DNA found in bacteria
201
Vector
Changed plasmid
202
Origin of replication function
Allows initiation of replication using host DNA polymerase
203
Antibiotic resistant gene function
Selectable marker
204
Promoter sequence
Drives expression of added gene
205
Screenable markers
Select cells that have taken up plasmid
206
Restriction sites
Allows ligation of gene of interest into the cloning vector
207
Ligation
Joining of two DNA strands or other molecules via phosphate ester linkage
208
Fluorescent activated cell sorting
Separating cells based on their fluorescent status
209
Restriction enzyme ligation
Cut and paste gene from nature. Restriction enzymes cut, DNA ligase pastes
210
5' exonuclease function
Chews back ends of DNA to make short single stranded overhangs
211
Reverse transcriptase function
Turns RNA to DNA
212
cDNA
DNA with introns removed
213
Signal peptide function
Ensures new proteins go to correct destination
214
Isoelectric focusing
Separates proteins based on pI
215
Phage display
Technique used to evolve antibodies
216
k₂
Catalytic rate constant - rate at which ES breaks down into products
217
DF =
V₂ / V₁
C₁ / C₂
218
Infrared wavelengths are shorter/longer
Longer
219
Monochromatic light
Light of a single wavelength
220
Beer's Law
Concentration of a solution is linearly proportional to the absorbance
221
Lambert's Law
The intensity of the transmitted light is linearly proportional to the path length
222
ℇ units
L.mol⁻¹ .cm⁻¹
223
ℇ defined as
Absorbance of 1 mol.L⁻¹ solution at a specific wavelength with light path of 1 cm
224
Standard curve (calibration curve)
Absorbance vs concentration at a particular wavelength
225
Function of an enzyme assay
Investigates how well an enzyme catalyses a reaction
226
PLP
Coenzyme in glycogen phosphorylase
227
BPG function
Reduces hemoglobins affinity for oxygen
Promotes release of oxygen in tissues where it is needed most
228
Which state does high pressure favour?
T state
229
Which state has a smaller volume?
T state
230
PCR
Amplifies DNA
231
pI > pH means group is
Positive
232
What are coding variants?
Changes in DNA sequence that can alter a genes protein product
233
What are transcription factor genes?
Genes that code for transcription factors
234
What are regulatory variants?
Changes in DNA sequence that change the regulation of the gene rather than the protein it codes for
235
FACS
Fluorescence activated cell sorting
BIOC192
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