MGD Flashcards
1
Q
What is the general structure of an amino acid?
A
NH2 - CH - R - COOH
2
Q
What groups are present in an ionised amino acid?
A
NH3+ & COO-
3
Q
Which amino acid isomer is found in nature?
A
L-isomer
4
Q
What are the classes of amino acids and give examples?
A
Non-polar amino acids - glycine, alanine, valine, leucine, proline, phenylalanine, isoleucine
Polar, uncharged - Cysteine, glutamine, tyrosine, asparagine, threonine, serine
Polar, charged - Lysine, arginine, histidine, aspartate, glutamate
5
Q
What is pK and how does it effect amino acids?
A
pK is the acid dissociation constant - how strong an acid is
if pH < pK = protonated
if pH > pK = deprotonated
6
Q
What are the characteristics of a peptide bond?
A
Rigid and planar and in trans conformation
7
Q
What is an isoelectric point?
A
The pH at which the protein has no overall net charge
8
Q
What is the secondary structure of a protein and what are the bonds involved?
A
Local spatial arrangement of polypeptide backbone
Covalent (peptide) bonds
Hydrogen bonds
9
Q
What is the tertiary structure of a protein and what are the bonds involved?
A
3D arrangement of all atoms in polypeptide Covalent (disulphide) Ionic Hydrogen bonds Van der Waals Hydrophobic
10
Q
What is the quaternary structure of a protein and what are the bonds involved?
A
3D arrangement of protein subunits Covalent (disulphide) Ionic Hydrogen bonds Van der Waals Hydrophobic
11
Q
Describe features of alpha-helix
A
3.6 amino acids per turn
0.54nm pitch
Right handed helix
R groups pointing outwards
Hydrogen bonds between C=O group and NH group 4 amino acids away
12
Q
What amino acids are strong helix formers and breakers?
A
Formers: Alanine and leucine (small)
Breakers: Proline (no rotation around N-C bond), Glycine (R=H which supports other conformations)
13
Q
Describe features of beta-sheet
A
Fully extended conformation
0.35nm between adjacent amino acids
R groups alternates between opposite sides of chain
Can be either parallel or antiparallel
14
Q
Describe the features of the two types of tertiary structure of a protein
A
Fibrous - single type of repeating secondary structure, long strands or sheets - support, shape and protection
Globular - several types of secondary structure, compact shape - catalysis, regulation
15
Q
Describe structure of collagen
A
Left handed triple helical arrangement of 3 alpha collagen polypeptide chains
Gly-X-Y repeating sequence
High degree of proline and hydroxyproline - Proline is correct geometry for extended alpha-chain conformation
Hydrogen bonds stabilise interactions between chains - due to hydroxyproline
16
Q
What causes protein denaturing and how does this occur?
A
Heat - increases vibrational energy
pH - alters ionisation states of amino acids
Detergents/organic solvents - disrupts hydrophobic interactions
17
Q
Describe the structure of heam
A
Protoporphyrin ring and Fe atom bound to 4 nitrogen atoms of the ring
Fe can bond to oxygen above the plane and histidine residue from protein on other side
18
Q
What are the features of myoglobin?
A
Compact single chain
75% alpha-helical
1 heam group
19
Q
What does binding of O2 to haem group do?
A
Pulls Fe into plane of ring changing from T state to R state
20
Q
Describe the oxygen binding curves for myoglobin and haemoglobin
A
Myoglobin - hyperbolic
Haemoglobin - sigmoidal (cooperative binding)
21
Q
Describe the structure of haemoglobin
A
2 polypeptide chains - alpha and beta
4 subunits, alpha 2, beta 2
Each chain has an associated heam group
Binding of oxygen causes a conformation change from T state to R state (oxygen binding promotes stability of R state)
22
Q
What is T state and R state?
A
T-state - Low affinity state
R-state - High affinity state
23
Q
Describe the role of 2,3-BPG
A
2,3-Bisphosphoglycerate - decreases affinity for O2, promoting release of oxygen in tissues
More present in high altitudes
24
Q
Describe the role of CO2 and H+ in oxygen binding to haemoglobin
A
Binding lowers affinity for oxygen —> The bohr effect.
Metabolically active tissues produce CO2 and H+, this moves the oxygen binding curve to the right meaning that more oxygen is given up at the site of the metabolically active tissues.
This ensures that delivery of O2 is coupled to demand.
25
Describe the difference between maternal and foetal heamoglobin
Maternal, HbA - alpha2-beta2
Foetal, HbF - alpha2-gamma2
HbF has a higher binding affinity for O2 than HbA which allows transfer of O2 to foetal blood supply from mother
26
What are the features of an active site
Occupies a small part of the enzyme
Formed by amino acids from different parts of primary sequence
Clefts or crevices
Complementary shape to substrate
Substrates are bound by weak multiple bonds
27
What is the Michaelis-Menten model for enzyme catalysis?
Proposes that a specific complex between the enzyme and the substrate is a necessary intermediate in catalysis. Predicts that plot will be rectangular hyperbola
Vo = Vmax [S] / Km [S]
Vmax = maximal rate when all enzyme active sites are saturated with substrate
Km = Substrate concentration gives 1/2 maximal velocity
28
What is the significance of Km
Low Km = high affinity
| High Km = low affinity
29
What is the lineweaver-burk plot?
```
Rearrangement of michaelis-menten equation
1/V0 = (Km / Vmax [S]) + 1/Vmax
Straight line
Y intercept = 1/Vmax
X intercept = -1/Km
```
30
Describe reversible enzyme inhibitors
Competitive - Binds at active site, affects Km not Vmax
| Non-competitive - Binds at another site on the enzyme, affects Vmax not Km
31
What are the methods of short term enzyme activity regulation
Substrate and product concentration - isoenzymes (different forms of same enzyme, different kinetic properties)
Enzyme concentration
Allosteric regulation - bind to allosteric binding site to either inhibit or activate (phosphofrucktokinase)
Covalent modification - phosphorylation
Proteolytic cleavage - Zymogens
32
Briefly describe blood clotting cascade
Two pathways that converge:
Intrinsic - damaged endothelial lining of blood cells promotes binding of factor XII - Ca2+ cofactor
Extrinsic - trauma releases tissue factor III
Converge to form factor X ---> prothrombin to thrombin activation ---> formation of fibrin clot from fibrinogen
33
Describe the role of carboxyglutamate (Gla) residues
The post-translational modification of blood clotting cascade factors and prothrombin in the ER of liver adds COOH groups into glutamate residues to form carboxyglutamate - requires vitamin K
Allows interaction within sites of damage through Ca2+ and brings together clotting factors
34
Describe formation of fibrinogen clot
Dancing crabs
2 sets of tripeptides - 3 globular domains linked by rods
Fibrinopeptides are removed by thrombin allowing polymerisation (globular C-terminal of beta and gamma with N-terminal of beta and alpha chains)
35
Describe the stopping of the clotting process
Dilution of clotting factors by blood flow
Digestion by proteases
Specific inhibitors (naturally occuring)
Plasminogen ---> Plasmin causes fibrin to be digested (proteolytic cleavage)
36
Describe DNA packaging
Histone octamer core with DNA strand wrapped round twice (nucleosome) linking histone cores
Loosely packed = beads on a string = euchromatin = genes expressed
Tightly packed = selenoid = heterochromatin = genes not expressed
37
What is the difference between RNA and DNA?
OH replaced for H on 2' carbon for DNA
38
How many rings does a purine have and what are the bases?
Two
| A and G
39
How many rings does a pyrimidine have and what are the bases?
One
| C, T and U
40
Which bases pair and how many bonds made between them?
```
A-T = 2 bonds
G-C = 3 bonds
```
41
How are nucleotides joined together?
Via phosophodiester bonds moving from 5' to 3'
42
Describe the cell cycle
```
Growth 1 = cell content replication
Cell cycle check point
S phase = DNA replication
Growth 2 = Double check and repair
Cell cycle check point
Mitosis = cell division
```
43
Describe DNA replication
Initiation - recognition of origin of replication, requires recruitment of DNA polymerase and other specific proteins, requires kick-start by primase
Elongation - DNA polymerase moves along both strands making a copy (consistent for leading strand, in segments for lagging strand - DNA ligase fills in gaps), DNA helicase unzips double strand
Termination - 2 replication ends meet to produce 4 strands
44
Describe the chromosome structure
Two chromatids joined together via centromere
| Centromere can be metacentric, submetacentric or acrocentric
45
Describe the phases of mitosis
Prophase - nuclear membrane disappears, spindle fibres form
Prometaphase - spindles attach to centromeres
Metaphase - chromosomes line up in centre
Anaphase - chromatids pulled to polar ends
Telophase - cells divide
46
How does meiosis differ from mitosis?
Two rounds of mitosis
Production of four non-identical cells
Germ line cells
47
What causes generation of genetic diversity?
Random assortment of chromosomes
| Crossing over of genetic material
48
Describe the difference between spermatogenesis and oogenesis
Spermatogenesis forms 4 sperm from one spermatocyte - takes 48 days
Oogenesis forms one mature ovum and 3 polar bodies - takes 12-50 years
49
What is co-dominance?
Two alleles are neither dominant over the other
| e.g. blood groups (A & B are co-dominant)
50
What is complementation?
More than one gene involved in producing phenotype
51
What is linkage and recombination?
Genes on same chromosome are linked
Genes on different chromosomes are not-linked
Recombination frequency between linked genes is dependent of the distance between the genes
52
What is a person's phenotype?
Physical characteristics or traits of genetic make up, genotype modified by environmental and developmental conditions
53
What is a person's genotype?
Genetic code it carries
54
Describe the process of transcription
Initiation:
1. Recognition of TATA box
2. Transcription factors bind
3. RNA polymerase binds to transcription factors
4. ATP is required
Elongation:
1. Replication of leading strand with nucleotides
Termination:
1. RNA polymerase reaches stop sequence
2. 5' capping by methylated guanine - prevents degradation
3. polyadenylation of 3' end
4. Splicing of introns to make mature mRNA
55
Describe the process of translation
Initiation:
1. 40S of rRNA binds to 5' cap
2. Methionine tRNA binds to 40S (amino acid for AUG, initiation codon)
3. ATP moves 40S and met-tRNA to initiation codon
4. 60S rRNA binds
Elongation:
1. rRNA moves along adding aminoacyl-tRNA with corresponding amino acid to codon which forms a peptide bond
2. Translocation (moves from A site to P site using GTP)
Termination:
1. Stop codon reached - no amino acid is added
2. Hydroxylation of last amino acid to form OH group
56
Describe the process of DNA gel electrophoresis
DNA fragments separated by size.
Placed on gel with cathode and anode, each DNA fragment moves towards positive end. The distance moved corresponds with size.
Requires: gel, buffer, power supply, stain
57
What is restriction analysis?
Uses restriction endonucleases (resitriction enzymes) that recognise specific DNA sequences then cut at that site to leave sticky ends.
It is possible to cut and paste DNA fragments together - DNA modification.
Useful for gene cloning - adding DNA into a plasmid and allowing bacteria to reproduce, extract the plasmid for analysis
58
Describe PCR
Polymerase chain reaction amplifying a small amount of DNA into large numbers of replications
3 steps:
1. DNA is heated to 95C to separate strands
2. Sample is cooled to 55C and sequence-specific primers anneal to target DNA
3. Sample is heated to 72C to allow DNA synthesis by Taq polymerase (thermostable)
59
Describe SDS-PAGE
Detergent SDS denatures protein molecules and masks the proteins charge which its large negative charge, allowing separate by molecular weight.
60
Describe the process of western blotting
SDS-PAGE followed by transferring the proteins to a membrane and specific proteins can be visualised using antibody binding
61
Describe ELISA
Enzyme-linked immunoabsorbant assays
1. Antibody of protein of interest is immobilised on solid support
2. Solution with protein of interest is added, antibody binds to protein, rest is washed away
3. Second antibody specific to protein applied with enzyme linked to it
4. Binding of second antibody measured by assaying for activity of enzyme linked to it
62
What is an enzyme assay?
Measuring enzyme activity by measuring substrate or product during reaction
63
Describe DNA sequencing
Determination of the nucleotide sequence using the dideoxy chain termination method
64
Describe southern blotting/hybridisation
Following DNA gel electrophoresis, fragments are transferred onto a solid membrane. A single-stranded DNA molecule labelled with radioactive or fluorescent tags used as a probe to find any complementary DNA sequences present on membrane
65
What is karyotyping?
Full set of stained metaphase chromosomes of an individual organised according to chromosome number
66
Describe FISH
Fluorescence in situ hybridisation
| Fluorescent probes for a specific genes or DNA stretches are used
67
What is a mutation?
A change in the genetic code either spontaneously or induced by mutagens creating a mutant allele or phenotype
68
What is a point mutation? What are the two subgroups? What are the three types?
Base subsititution either a transition (purine to purine, pyrimidine to pyrimidine) or transversion (purine to pyrimidine)
They can be:
1. Silent mutation - no change to the amino acid
2. Missense mutation - replaces one amino acid for another
3. Nonsense mutation - changes amino acid for stop codon
Point mutations outside non-coding regions can cause change to protein binding sites, promoter sequences, splice sites etc
69
Describe insertion or deletion with regards to mutations
A sequence that is either added or deleted, can be a single nucleotide up to millions of nucleotides long (tandem duplications).
They can cause a frame shift mutation if the number is not a multiple of 3 or no change in the reading frame but change in amino acid code if multiple of 3
70
Describe DNA repair
Mismatch repair - post-replication process in which mismatched based is changed for the correct one
Excision repair:
1. Base-excision repair - single nucleotide due to oxidation, alkylation, hydrolysis or demaination
2. Nucleotide excision repair - repairs damage affecting 2-30 nucleotides caused by external agents e.g. chemical mutagens, UV, ROS
71
What is normal karyotype nomenclature for a male and female?
Male - 46,XY
| Female - 46,XX
72
Name the types of numerical chromosomal abnormailities
Polypoidy - multiple numbers of all chromosomes
| Aneuploidy - multiple copies of one chromosome
73
Name the types of structural chromosomal abnormailities
Balanced - the exchange rearrangement of genetic material does not cause any missing or extra genetic information - inversion
Unbalanced - the structural chromosomal changes cause missing or extra genetic information - deletion, duplication
74
What is an isochormosome?
Creation of two non-identical chromosomes, one is both p arms and other is both q arms
75
What is translocation chromosome mutation?
Rearrangement of genetic material to a non-homologous chromosome - no loss of genetic material
76
What is reciprocal translocation?
Exchange of genetic material between two non-homologous chromosomes - no loss of genetic material
77
What is Robertsonian translocation?
Rearrangement of genetic material between 2 chromosomes - q arms of acrocentric chromosomes combine to from one super-chromosome with loss of both p arms
78
Describe collagen synthesis
C - Cleavage of signal peptide
H - Hydroxylation of proline and lysine residues
A - Addition of N-linked oligosacchardies & galactose to hydroxylysine
D - Disulphide bonds form between aligned chains
P - Procollagen formed (triple helix)
O - O-linked glycosylation
G - Transport vesicle in golgi and exocytosis
R - Removal of terminal peptides (procollagen peptidase)
L - Lateral aggregation to form fibrils
80
What are the components of a signal sequence?
It is located at the N-terminus of a protein
Contains:
1. Stretch of ~10-15 hydrophobic residues
2. 1 or more positively charged residues near amino terminus before hydrophobic sequence
3. Few polar amino acids within C-terminal region of signal sequence
81
Describe the protein secretion pathway
1. Protein synthesis initiated on free ribosomes & N-terminal signal sequence produced
2. Signal sequence recognised by signal recognition particle (SRP)
3. GTP-bound SRP directs ribosome to SRP receptor on ER
4. SRP dissociates with hydrolysis of GTP - translocon opens
5. Signal sequence cleaved by signal peptidase
6. Protein synthesis continues
7. Ribosome dissociates - translocon closes
8. Protein folds
82
Describe synthesis of insulin
Preproinsulin synthesised into the ER where signal sequence is cleaved (by sign 8al peptidase) and disulphide bonds are formed
Transported into Golgi where C peptide is cleaved by endopeptidases to give insulin (active)
85
What modification occur in the ER?
Correct folding
Disulphide bonds formed
N-linked glycosylation
87
How does genetic variation occur?
Random assortment of chromosomes during metaphase and crossing over
88
What is N-linked glycosylation?
Sugars added to amine group on asparagine via N-linked glycosyl link
The sugars are assembled on a membrane lipid carrier and then transferred
89
What modification occur in the Golgi?
O-linked glycosylation
90
What is O-linked glycosylation?
Sugars added to hydroxyl of serene and threonine residues
| Glycosyl transferase builds up sugar chain and attaches it via a glycosidic link
91
What are the two types of intracellular secretion?
Constitutive secretion - continuous process, proteins packaged into vesicles and released continuously by exocytosis e.g. Serum albumin and collagen
Regulated secretion - proteins released in response to a signal (hormone), proteins packaged into vesicles but not released until stimulus received e.g. Insulin
92
How is a protein targeted to the ER?
N-terminus signal that is removed - made of 6-12 hydrophobic amino acids & 1 or more basic amino acids
93
How is a protein targeted to the mitochondria?
N-terminus signal that is removed - made of amphipathic helix 920-50) residues
94
How is a protein targeted to a peroxisome?
C-terminus signal that remains - serine-lysine-leucine at C terminus
95
How is a protein targeted to the nucleus?
Internal signal that is not removed - cluster of basic amino acids
96
Describe the process by which a newly synthesised protein enters the mitochondria
1. Chaperone keeps protein unfolded
2. Signal binds to receptor
3. Protein fed through pore in membrane
4. Proteins moves through pore in inner membrane
5. Targeting signal cleaved
97
Describe the process by which a newly synthesised protein enters the peroxisome
1. PTS binds to cargo
2. Binds to peroxisomal import receptor
3. Peroxisomal import receptor merges into the translocon
4. PTS cargo complex dissociates in the cell and peroxisomal import receptor dissociates extracelllularlly
98
Describe the process by which a newly synthesised protein enters the nucleus
1. Importin binds to protein with nuclear localisation signal (NLS)
2. Transported across membrane
3. Ran-GTP binds inducing a conformational change
4. Protein is displaced intracellularly
99
Describe allosteric regulation
Sigmoidal relationship between rate and substrate concentration
Substrate binds making subsequent binding either easier or harder - R & T state
It binds to an allosteric binding site
Activator increase R state
Inhibitors increase T state
