1
Q
Protein length
A
50-100 Å
2
Q
1 Å =
A
10⁻¹⁰ m
3
Q
Proteins start with _____ terminus
A
Amino
4
Q
Three ways to determine protein structure
A
- Protein crystallography
- Cyro-electron microscopy
- NMR spectroscopy
5
Q
Lower quality way to determine protein structure
A
NMR spectroscopy
6
Q
Trypsin
A
Enzyme that breaks down proteins in digestion
7
Q
Amylase found in
A
Saliva
Pancreatic juices
8
Q
Amylase
A
Breaks down starch into sugar
9
Q
Kinase
A
Adds phosphate
10
Q
Spike protein found
A
On the surface of some enveloped viruses
11
Q
Spike protein function
A
Allows virus to enter host cells
12
Q
DNA polymerase function
A
DNA replication
13
Q
RNA polymerase function
A
Creates strand of RNA that is complementary to a strand of duplex DNA
14
Q
Duplex DNA
A
Double strand DNA
15
Q
Amino acid that is non-chiral
A
Glycine
16
Q
Amino acids that can absorb UV light
A
Tryptophan
Tyrosine
Phenylalanine
17
Q
Negatively charged side chains are basic/acidic
A
Acidic
18
Q
Pka = pH when group is ___% ionised
A
50%
19
Q
pH is pI when net charge is
A
0
20
Q
RNA -> proteins occurs at
A
Ribosome
21
Q
Phosphatases
A
Remove phosphate groups via hydrolysis
22
Q
Hydroxylation often has ________ involved
A
Proline
Lysine
23
Q
Carboxylation often has _________ involved
A
Glutamate
24
Q
Glycosylation often has ________ involved
A
Threonine
Asparagine
25
Peptide bond has ___% double bond character
40%
26
Peptide bond has rotational barrier of ___ kJ/mol
80 kJ/mol
27
Rotational barrier
Energy difference between the most and least stable conformations when a chemical rotates around a bond
28
Polypeptide = ___ amino acids
>50
29
ɸ
Phi
30
ψ
Psi
31
Bond angle between N and Cₐ
Phi
32
bond angle between Cₐ and C'
Psi
33
Psi and Phi bond angles can be between
-180° and +180°
34
Chain angle between C' and N notated by
ω (omega)
35
Alpha helix is left/right handed
Right
36
Phi rotations lead to ________ collisions
O-O
37
Psi rotations lead to ________ collisions
NH-NH
38
Peptide bond is ______ Å shorter/longer than Cₐ- N
0.13
Shorter
39
Trans peptide bond angle
~ 180°
40
Cis peptide bond angle
~ 0°
41
Alpha helix hydrogen bonding between O-N distance is
~ 2.9 Å
42
Alpha helices have ___ residues per turn
3.6
43
Hydrogen bonds have energy of
12-18 kJ/mol
44
Alpha helices have rise of ____ per turn
5.4 Å
45
d refers to
Distance between n and n+4
46
d = ____ per residue
1.5 Å
47
In alpha helices phi =
~ -57°
48
In alpha helices psi =
~ -47°
49
Helix breakers
Amino acids that disrupt the formation of alpha helices
50
Eg. of helix breakers
Glycine
Proline
51
In alpha helices, each amino acid side chain is separated by ___°
100°
52
____ strands per sheet
2-10
53
Average strand length
6 amino acid residues
54
Maximum strand length
15 amino acid residues
55
Turns have ____ residues
3-4
56
Almost ___% of protein residues are in turns
30%
57
Turns involve a lot of
Glycine
Proline
58
Type 1 turns
Hydrogen bond is between residues n and n+3
59
Type 2 turns
Hydrogen bond is between residues n and n+2
60
More common type of turn
Type 1
61
Supersecondary structure
Elements of secondary structure that are connected by turns, loops or coils
62
Helix-turn-helix commonly found in
DNA binding proteins
63
Helix-loop-helix is a
Calcium binding protein
64
Beta-hairpins
Two antiparallel beta strands next to each other connected by a turn
65
Amphipathic
Has both hydrophilic and hydrophobic parts
66
Alpha-domain family
Amphipathic helices with side chains in hydrophobic core
67
Most common in alpha-domain family
Four helix bundle
68
Helices like to sit ___° away from each other
20-50°
69
Alpha/beta family example
Alpha/beta horseshoe fold
70
Chaperone independent
Polypeptide is made and folds up
71
Chaperone dependent
Chaperone steps in
72
Chaperonin dependent
Super-chaperone steps in
73
Chaperone independent and chaperone dependent make up ___% of of protein folding
85%
74
Chaperonin dependent makes up ___% of protein folding
15%
75
Prions (PrP)
Proteins that cause misfolding of normal versions of the protein
76
G‡
Gibbs activation energy
77
Oxidoreductases
Redox enzymes
78
Transferases
Enzymes that transfer functional groups
79
Hydrolases
Enzymes that do hydrolysis
80
Lyases
Enzymes that do non-hydrolic breaking/making of bonds
81
Isomerases
Enzymes that transfer substrate to an isomeric form
82
Ligases
Enzymes that join two molecules together, usually coupled to ATP cleavage
83
Two classes of cofactor
Metal ions
Coenzymes
84
Activation energy is lowered by
Destabilising ground state
Stabilising transition state
85
Catalytic mechanisms
Proximity and orientation effects
Enzyme/chemistry specific
86
Enzyme/chemistry specific catalytic mechanisms
Acid-base catalysis
Metal ion catalysis
Covalent catalysis
87
First linear part of graph is ________ order kinetics so rate does/doesn't depend on [S]
First order
Does
88
Second part of graph is _______ order kinetics, so rate does/doesn't depend on [S]
Zero order
Doesn't
89
Two kinetic parameters from a V vs [S] graph
Km
Vmax
90
Vmax
Maximum velocity when [S] = ∞
91
Km
[S] needed to reach half of Vmax
92
Haldane's steady state assumption
The rate of ES formation = rate of it's breakdown
93
E + S ⇌ ES
Slow or fast?
Fast
94
ES → E + P
Fast or slow?
Slow
95
Michaelis constant
Km
96
Low Km means
High affinity between E and S
97
ES dissociation constant
Km = K₋₁/K₁
98
In Lineweaver-Burk plot, y intercept =
1/Vmax
99
In Lineweaver-Burk plot, x intercept =
-1/Km
100
Good enzyme has ____ kcat and _____ Km
High kcat, low Km
101
kcat
Number of substrate molecules converted to product per unit of time when E is saturated with S
102
kcat =
= k₂
= Vmax/[E]ₜ
103
Overall measure of enzyme efficiency
kcat/Km
104
Diffusion controlled limit
When reaction is so fast the rate is determined by the rate at which E and S diffuse together
105
Absolute upper limit for enzymes
~ 10⁹ s⁻¹ M⁻¹
106
Enzymes with kcat/Km above ____ are perfect catalysts
10⁸ s⁻¹ M⁻¹
107
Consequence of competitive inhibition
Slows reaction down
108
In competitive, Vmax decreases/stays the same and Km increases/stays the same
Vmax stays the same
Km increases
109
In non-competitive, Vmax decreases/stays the same and Km increases/stays the same
Vmax decreases
Km stays the same
110
In mixed inhibitors, Vmax decreases/stays the same and Km changes/stays the same
Vmax decreases
Km changes
111
Feedforward regulation
A molecule activates an enzyme further down the pathway
112
Feedback regulation
A product regulates the activity of an enzyme earlier in the pathway
113
Allostery
A molecule binding at additional sites
114
Proteolytic cleavage
Made in longer form, cleaved when used
115
Solubility of oxygen in saline
0.2 mmol L⁻¹
116
Oxygen is stored in muscle in
Myoglobin
117
Oxygen in muscle is gone in
7 seconds
118
Solubility of oxygen in muscle is
0.5 - 0.7 mmol L⁻¹
119
Myoglobin primary structure
~ 150 amino acids
120
Myoglobin secondary structure
8 alpha helices (labelled A-H) connected by loops
121
Heme interacts with which amino acid in myoglobin?
Histidine F8
122
In myoglobin, the six Fe coordinate bonds go to
4 - N atoms of heme
1 - N from histidine F8
1 - oxygen
123
What gives myoglobin its red colour?
Molecular electronic orbitals of the heme
124
Oxygenated/deoxygenated myoglobin gives two peaks on a spectroscopy graph (as opposed to one)
Oxygenated
125
Oxygenated/deoxygenated myoglobin is bright red
Oxygenated
126
Quaternary structure of hemoglobin
Non-covalent tetramer
127
Does myoglobin or hemoglobin bind oxygen more weakly?
Hemoglobin
128
Partial pressure of oxygen in lungs
~100 Torr
129
Partial pressure of oxygen in resting muscle
~20 Torr
130
What shape is a myoglobin oxygen saturation curve?
Hyperbolic
131
What shape is a hemoglobin oxygen saturation curve?
Sigmoidal
132
Which becomes saturated with oxygen at high concs?
Hemoglobin
133
Cooperativity
When one oxygen binds to a heme, the affinity of the other three increases
134
Cooperative behaviour = _______ curve
Sigmoidal
135
T state and R state apply to myoglobin/hemoglobin
Hemoglobin
136
T state = _____ affinity for oxygen
Low
137
R state = _____ affinity for oxygen
High
138
Hemoglobin is predominantly in ___ state
R state
139
Where does BPG bind to hemoglobin?
Allosteric site between beta-chains
140
Lower pH favours ___ state
T state
141
How does CO₂ stabilise T state?
Lowering pH
Binds to amino terminus
142
Beta hemoglobin is expressed
After baby is born
143
Alpha hemoglobin is expressed
Always
144
Gamma hemoglobin is expressed
Only while in womb
145
Foetal hemoglobin holds oxygen weaker/tighter
Tighter
146
Foetal hemoglobin is less/more sensitive to BPG
Less
147
HbS
Abnormal type of hemoglobin that can cause sickle cell anemia
148
HbS variant
E6V
In beta chain
149
Warfarin low doses effect
Prevents blood clots post stroke or heart attack
150
Which has only one active site, receptors or enzymes?
Enzymes
151
Endogenous ligands are usually
Agonists
152
Synaptic
Really close
153
Paracrine
Acts on nearby cells
154
Endocrine
Released in one space, picked up in another
155
Phosphodiesterases
Break down cAMP and cGMP
156
Gɑₛ
Stimulatory G protein
157
Gɑᵢ
Inhibitory G protein
158
Gɑₛ and Gɑᵢ effect
Adenylate cyclase
159
Adenylate cyclase function
Makes cAMP
160
When GDP is bound to G protein, it is
Inactive
161
When GTP is bound to G protein, it is
Active
162
GTP replaced GDP when
G protein interacts with receptor
163
What hydrolyses GTP to GDP?
GTPase
164
How do RTKs start signal transduction?
Phosphorylation of adaptor proteins
165
Transcription
DNA → RNA
166
Translation
RNA → Protein
167
Transcribed region
Part of gene that is copied into mRNA
168
Introns and exons found where?
Transcribed region
169
RNA polymerase function
Makes mRNA
170
DNA has __ base where RNA has __ base
DNA = T
RNA = U
171
Where is mRNA made?
Nucleus
172
Where is mRNA translated?
Cytoplasm
173
Function of promoter region
Controls gene transcription
174
Promoter region contains
Transcription factor binding sites
RNA polymerase binding site
175
What are binding sites in promoter region?
Short specific DNA sequences
176
Transcription factor function
Binds to promoter and flags down RNA polymerase
177
Two kinds of transcription factors
Activators
Repressors
178
How many cells in the human body?
~30 trillion
179
How many different types of cells in the human body?
~200
180
How many protein coding genes?
~21,000
181
How many proteins can be made?
~85,000
182
Alternative splicing
Exons spliced in different combinations
183
How many genes does each cell type express?
~11,000 - ~17,000
184
How many genes are expressed in all cells?
~10,000
185
How many genes are expressed only in specific cells?
~1,000 - ~2,000
186
Ribosomes made of
rRNA and proteins
187
What occurs in ribosomes?
Translation
188
C always paired with
G
189
A always paired with
T or U
190
tRNA function
Reads codons in mRNA
191
tRNA structure
Anti-codon on one end, amino acid on the other
192
Anti-codon
Complementary sequence to mRNA codon
193
Three stages of translation
1. Initiation
2. Elongation
3. Termination
194
Initiation
Ribosome, mRNA and first tRNA come together to form translation initiation complex
195
First tRNA always codes for
Methionine
196
Elongation
Ribosome moves along mRNA adding amino acids
197
Termination
Stop codon causes translation complex to break apart
198
How many possible codons?
4³ = 64
199
Which amino acids can't be coded for by multiple codons?
Methionine
Tryptophan
200
Codon redundancy
Most amino acids are specified by more than one mRNA codon
201
Humans are ___% different from each other
0.1%
202
How many base pairs is the whole (diploid) human genome is made up of?
6 billion
203
Alleles
Different forms of a variant in the same location
204
Single nucleotide polymorphism
Single base change in the DNA sequence
205
Insertion-deletion
Addition/removal of one or more bases
206
Where are coding variants located?
Exon
207
List non-coding variants
Intergenic variant
Regulatory variant
Intron variants
208
Where are intergenic variants located?
Stretch of DNA between two genes
209
Where are regulatory variants located?
Promotor region
210
Non-coding variants can change
Expression of gene
211
Coding variants can change
Amino acid sequence and potentially function of protein
212
Synonymous
Altered codon specifies for the same amino acid
213
Missense
Altered codon specifies for one different amino acid
214
Nonsense
Altered codon specifies early stop
215
Inframe insertion/deletion
Gain/loss of one amino acid
All codons after variant read correctly
216
Frameshift insertion/deletion
Codons after variant read incorrectly
217
Diploid
Cells contain two complete sets of chromosomes
218
Homologous pair
Two of each chromosome that diploid organisms have
219
Phenotype
Observable trait
220
Genotype
Combination of alleles a person has for a particular genetic variant
221
Homozygote dominant
Double dominant
222
Heterozygote dominant
One of each
223
Gain of function allele often shown in which inheritance pattern?
Dominant
224
Genotyping
Determining the specific genetic alleles in an organism's DNA
225
PCR primer
Small sequence of DNA complementary to specific sequence surrounding DNA of interest
226
What does PCR-RFLP use to determine genotype?
PCR
Restriction enzymes
Gel electrophoresis
227
Restriction enzymes
Proteins isolated from bacteria that cut DNA
228
Monogenic diseases affect ___% of people
6%
229
Proto-oncogenes
Normal cells that regulate cell growth and division
230
Oncogene mutation is _____-of-function
Gain
231
___% of cancers caused by inherited genetic variants
10%
232
Inherited genetic variants require less/more mutations to accumulate before cancer develops
Less
233
Recombinant DNA technologies
Joining bits of DNA
234
Reporter gene function
Produces a signal to monitor gene expression
235
Plasmid
Circular piece of of double strand DNA commonly found in bacteria
236
Vector
Changed plasmid
237
Origin of replication function
Allows initiation of replication using host DNA polymerase
238
Antibiotic resistant gene
Provides survival advantage
Selectable marker
239
Promotor sequence
Drives expression of added gene
240
Screenable markers
Select cells that have taken up plasmid
241
Restriction sites
Allows ligation of gene of interest into the cloning vector
242
Ligation
Joining of two DNA strands or other molecules via a phosphate ester linkage
243
Fluorescent activated cell sorting
Separating cells based on their fluorescent status
244
Restriction enzyme ligation
Cut and paste gene from nature. Restriction enzymes cut, DNA ligase pastes
245
Prefer straight/zig-zag cutting restriction enzymes
Zig-zag
246
DNA ligase function
Forms phosphodiester bonds between DNA molecules
247
5' exonuclease function
Chews back ends of DNA to make short single stranded overhangs
248
Reverse transcriptase function
Turns RNA into DNA
249
cDNA
DNA with introns removed
250
Signal peptide function
Ensures new proteins go to correct destination
251
Isoelectric focusing
Separates proteins based on pI
252
Phage display
Technique used to evolve antibodies
253
k₂
Catalytic rate constant
Rate at which ES breaks down into products
254
DF =
V₂ / V₁
C₁ / C₂
255
Infrared wavelengths are shorter/longer
Longer
256
Absorbance maxima
Wavelength that is absorbed by a molecule more than any other wavelength
257
Monochromatic light
Light of a single wavelength
258
Beer's Law
Concentration of a solution is linearly proportional to the absorbance of that solution
259
Lambert's Law
The intensity of the transmitted light is linearly proportional to the path length
260
Absorbance (A) =
ℇ x c x l
261
ℇ units
L.mol⁻¹ .cm⁻¹
262
ℇ defined as
Absorbance of 1 mol.L⁻¹ solution at a specific wavelength with light path of 1 cm
263
Standard curve (calibration curve)
Absorbance vs concentration at particular wavelength
264
Function of an enzyme assay
Investigates how well an enzyme catalyses a reaction
265
PLP is a
Coenzyme in glycogen phosphorylase
266
Hexokinase function
Catalyses phosphorylation of glucose
267
BPG function
Reduced hemoglobins affinity for oxygen
Promotes release of oxygen in tissues where it's needed most
BIOC192
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