Part 1 Flashcards
1
Q
What is protein folding and missfolding?
A
Folding= Acquisition of the correct structure of preform designated functions Missfolding = the failure to acquire the correct structure hence the failure to function
2
Q
How do we depict protein structure if even one aa is a complex structure?
A
Alanine= 12 atoms
Protein consists of 20 different aa and average 500 aa
simplifying structure allows scientists to understand how mutations affect cell function
2 major protein structures- Alpha and beta
3
Q
What are the different representations of alpha helix?
A
- Backbone
- Sticks
- Space filling
- Ribbon
Same for b sheet
4
Q
Beta representations
A
Sheet- flattened arrows pointing to the c terminus= Anti-parallel
H bonds between polypeptide chains lying parallel- adjacent region to beta sheet
Loop between b sheets provides structure to protein
5
Q
What is the alpha heical structure?
A
A spiral conformation in which every backbone N-H group donates a H bond to the backbone C=O group which of aa located approx 4 residues earlier along protein structure
7 transmembrane helical receptor
6
Q
What is the structure of beta helical sheet?
A
Beta sheets consist of b strands connected laterally by backbone hydrogen bonds
Different conditions consist of different bonding type
Aromatic residues often found in b strands in the middle of b sheets
7
Q
What interactions hold protein structures together?
A
Hydrogen bonds= Hold a shared H between O and N atoms
Ionic bonds= attraction between +ve and -ve charged ions
Weak van der walls= short range hydrophobic interactions
Disulfide bonds= involve a chemical link between 2 adjacent cystines
8
Q
Folding of polypeptide chain in aqueous environment
A
Inside= Hydrophobic- Non polar side chains contained Outside= Hydrophillic- Polar side chains form H bonds with water
9
Q
What is the quaternary structure?
A
Relationship between individual proteins in a multimeric complex (often duplication and multimersiation occurs)
Protein said to contain 2 Alpha and 2 beta
10
Q
What methods can be used to sequence aa directly?
A
Edman degradation- each aa has own MW remove sequence of a, react peptides using PIT- label amino-terminal residue without disrupting aa bonds
Mass spectroscopy
11
Q
How can secondary structure be predicted?
A
From primary by either de nova (the beginning) or alignment with other proteins of known structure using protein device- hydrophobic interacts with hydrophobic so align next to each other
12
Q
What is coil coil?
A
When 2 alpha helix wrap around each other to form helical structure Amphatic side (leu and val) pi Triple coil- 3 helices twist around central aspect
13
Q
What are the several techniques to obtain ‘real’ protein structure?
A
Circular Dichroism (CD)
- circularly polarising (190-250nm), give characteristic shape of CD
- different temps show stability- less stable faster lose CD characteristic
- cooling shows if it will refold
X-ray Crystallography
- x ray beam fired through protein crystal, most goes through few deflected
- structure traced from defraction
Nuclear Magnetic Resonance (NMR)
- Calmodium- binds activating proteins/kinases
- protein of interest= highly pure and labelled with isotopes
- C13 and N15 wobble spin
- Introduce into proteins recombinantly (grown on media)
Electron Microscopy
- Uses negative stain on actin filaments
- dried on grid, lightly coated with heavy metal
- see deposits of metal around protein synthesis
Cryo-electron microscopy
- liquid nitrogen to freeze
*For all these techniques you need to purify first
14
Q
Pros and cons of various structural techniques
A
CD- cheap, quick, no size limit, low resolution but limited dynamics
X-ray- costly, crystals slow, data fast, high resolution, no size limit but bigger is harder, some artefacts, limited dynamics
NMR- costly, data fast, analysis slow, reasonable resolution, size 50KDA, good dynamics, repetitive process
EM- costly, medium speed, good resolution, no size limit, no dynamics
15
Q
Where is most eukaryotic DNA is contained in?
A
Nuclear chromosones
16
Q
Nuclear chromosomes comprises linear DNA and proteins which have specialised functions, these are:
A
- Packaging and unfolding of DNA repair and genetic recombination
- controlling DNA replication, DNA repair and genetic recombination
- maintaining chromosome integrity by preventing loss of end sequence
- governing proper chromosome segregation during cell division
- Regulating gene expression
- Mitochondria and chlorplast also contain small, circular chromosome
17
Q
What are diphoid eukaryotic cells?
A
Contain two copies of each chromosome
18
Q
Chromosome pairs differ by?
A
Size
DNA sequence content
19
Q
During interphase which chromosomes occupy distinct sub nuclear territories?
A
The nuclear periphery is composed of transcriptionally inactive DNA
Inside= active
Transcriptional activation of gene accomplished by movement from periphery to centre of nucleus
20
Q
Which fibre highly coats chromosomes?
A
chromatin
21
Q
Biochemical anaylsis of nucleosomes reveals a protein core around which DNA wound, like cotton on a bobbin
What info do we know?
A
The protein subunits of the nucleosomes are core histones
The N terminal tails of the 8 core histone subunits project out from the nucleosome core and are free to interact with other proteins, facilitating regulation of chromatin structure
22
Q
What are linker histones?
A
Example H1
Strap DNA onto histone octamers and limit movement of DNA relative to the histone octomer
Stabalizes formation of 30nm fibre
23
Q
What is DNA packaged by?
A
Histone octomers
Packaged into a compact, flexible 30nm chromatin scaffold then can be remodelled to accommodate protein complexes involved in gene transcription and DNA replication
24
Q
What is a telomere?
A
DNA sequence at ends of linear chromosome maintain chromosome integrity- stops it getting shorter
Repeat array eg TTAGGG are synthesised by telomere
Define chromosome ends to maintain identity
25
What is the replication origin?
DNA sequence where DNA replication is initated
26
What is the centromere?
DNA sequence on which kinetochore assembles and mediates chromosome segregation at mitosis and meiosis
contain specialised proteins and DNA sequences
Contain alpha- satelite DNA repeats
27
What is the kinetochore?
Protein complex that binds microtubules in the mitotic spindle
28
What are the different plates of the kinetchore?
Inner- bind to alpha satellite DNA
| Outer- binds protein components of mitotic spindle
29
What is centromeric chromatin?
Contains specialised histones that mediate attachment of the chromosome to the kinetochore and modified histones that help to hold sister chromatids together
30
What is the centromere in yeast like?
Centromere is a basket that links a single nucelosome of centrameric chromatin to a single microtubule (not big array)
31
What percentage of the DNA sequence of eukaryotic genome encodes for cellular proteins?
1.5%
32
What percentage of the human genome is made up of repeated DNA sequence elements?
50%
33
What are the 3 different types of transposons (mobile genetic element that relocate across the genome)?
1. DNA transposons- move by cut and paste without self duplication eg. chromosome A-B, powerful mutant
2. Retroviral retrotransposon- Replicate via RNA intermediate, producing new DNA copies that integrate new genomic location using self-encoded reverse transcriptase.
3. Non-retroviral polyA retrotransposons- in vertebrae genomes and replicates via an RNA intermediate using its own retrotransposon encoded reverse trranscriptase- copy and paste
34
Non retroviral polyA retrotransposon sequence
1. L1 in chromosome
2. L1 RNA synthesis
3. Reverse transcriptase binds L1 RNA
4. Cleavage of first strand of target DNA
5. DNA primed reverse transcriptase
6. 2nd strand produced
7. L1 DNA copy of new position @genome
35
What is DNA replication?
Semi conservative
36
DNA synthesis occurs in which direction?
5' to 3'
| By formation of phosphodiester bonds- formed by hydroxyl grouo carrying out a neuclophillic attack
37
Why is DNA synthesis effectively an irreversible reaction?
It is coupled to breakdown of PPi to 2pi by pyrophosphatase.
The free energy required for DNA synthesis is provided by the breakdown of 2 high energy phosphate bonds
dNTP + (dNMP)n - (dNMP)N+1 + 2Pi
38
How does it go from triophosphate to pyrophosphate?
Incoming deoxyribonucleoside triphosphate in the 5' to 3' direction of chain growth
39
What is the direction of DNA synthesis and what does this mean?
DNA synthesis can only proceed 5' to 3' direction and the DNA strands are oriented antiparallel
This means that both strands cannot be synthesised continuously
Template strands for leading strands and lagging strands synthesis are orientated antiparallel to one another at the replication fork
40
What are the leading and lagging strand?
```
Leading= continuous 5' to 3'
Lagging= discontinuous 5' to 3' contains okazaki fragments from breaks in replication
```
41
What is all DNA synthesis initiated by?
Extension of a short primer or RNA
Short primer is synthesised by DNA primase and only requires a DNA template and NTPs
RNA initiates DNA synthesis
42
What does the lagging strand require?
1. DNA primase- makes RNA primer
2. DNA polyermase- extends RNA primer, requires primer template junction
3. ribonuclease H- removes RNA primer
4. DNA polymerase- extends across gap
5. DNA ligase-seals the nick- to convert Okazok fragments into a continuous strand of DNA
43
How does DNA ligase work?
Uses the energy of ATP hydrolysis to ligate newly synthesised, adjacent DNA fragments in a 2 step catalytic reaction
ATP+ 5-P - P-P + 5-P AMP
P-P - 2pi + free energy
44
Why is the ligand process energetically favourable?
Bc of conversion of ppi and zpi by pyrophasphatase- coupled reaction
strong and stable
45
What does DNA helicase do?
Uses ATP to separate parental DNA strands at the replication fork and move the replication fork forward= rotational movement
46
Mutations encoded for a DNA helicase
Cause human disease
- Werner syndrome- a progenia (premature ageing)
- Bloom syndrome - rare cancer syndrome caused by loss of function mutations in the RecQ family DNA helicase which maintains genome identity
47
What is processitivity?
The number of nucleotides that a polymerase can incorporate into DNA during a single template‐binding event, before dissociating from a DNA template
DNA synthesis increases when the processitivity of the polymerase increases
*polyermase recruited to replication fork- tendency to extend @ 3' is greater then fall off
48
What happens once the first step in DNA synthesis is done?
Interaction of enzyme with the primer
| Template junction is maintained and addition of further nucleotide are very rapid
49
What is it enhanced by?
The sliding clamp and clamper loader- sliding clamp hold DNA polymerase on the DNA
50
How does the sliding clamp work?
Sliding clamp and clamp loader come together
Atp binding to clamp loader opens sliding clamp
DNA engaged in clamp
ATP hydrolysis locks sliding clamp around DNA and releases clamp loader
DNA polymerase binds to sliding clamp
51
The sliding clamp
Encircles the DNA like a nut and bolt and helps to move DNA polyermase forward
Human sliding clamp, proliferating cell nuclear antigen (PCNA) has a near identical 3D structure to this E.coli protein
52
What are single stranded binding proteins?
SSBCs expose single stranded DNA in the replication fork, making it available for templating synthesis of the new strand and easing replication fork progression
Keep replication fork open and enhance processitivity of DNA polymerase
53
What are hairpins?
Single stranded region of DNA template with short regions of base pairs
54
What are DNA topisomerases?
Prevent DNA from becoming tangled during DNA replication
- Unwinding of parental DNA strands at the replication fork introduces superhelical tension into the DNA helix
- Tension is relaxed by DNA topisomerases which nick and reseal backbone of helix
55
What are the different types of Nick and reseal?
Type 1= Nick and reseal one of the 2 DNA strands, no ATP required
Type 2= Nick and reseal both DNA strands, ATP required
56
Control of DNA replication- Initiation at replication origin
Specific DNA sequences (origins) direct the initation of DNA replication by recruiting initiator proteins
examples
1. Yeast- autonomously replicating sequence (APS)
2. Human- LMNB2, MHC, HB- defined by chromosome
57
Initiation of DNA replication in eukaryotes is biphasic:
1. Replication selection= formation of a pre-replicative complex occurs in G1
2. Origin activation= Unwinding of DNA and recruitment of DNA
polyermase occurs in s phase
Together ensure that each chromosome is only replicated once per cycle
58
Steps of pre-replicative complex
1. ORC (Origin recognition complex)- binds to replicator sequence
2. Cdt1 +Cdc6- helicase binding proteins bind to ORC
3. mcm2-7- Binds to complete formation of pre-RC
59
How does high cyclin-dependent kinase (cdk) activity affect s phase?
Activates existing pre-RC but prevents formation of new Pre-RCs
60
How does low cdk activity affect in G1 phase?
Pre-RC formation allowed
No pre-RC activation
No pre-RC formation in G2 and M
61
How is DNA replication finished?
Using an RNA primer to start means an end replication problem for linear eukaryotic chromosomes .
Ribonuclease H removes RNA primer, DNA polyermase and ligase close all but one gap-Okazaki fragment- incompletely replicated DNA
End sequence lost
62
What happens in the next round of replication of a DNA molecule (without a telomere)?
Ribonuclease H removes RNA primers, further shortening the newly synthesised DNA strands at the 5' end
RNA primer removal causes progressive shortening of linear DNA sequence
The addition of TTAGGG repeats by telomerase compensates for the loss of telomere sequences causedby RNA primer removal and prevents chromosome shortening
63
What is the extended 3' end DNA now enough to enable?
DNA primase to bind and initiate new RNA primer synthesis which can then be extended as an extra okazaki fragment
64
What does telemerase do?
Telomerase contains an RNA component that specifies telomere sequence
Telomerase is a ribonucleoprotein with an intrinsic RNA component that acts as a template on which telomere repeat sequences are synthesised in a step-wise process= the telomere shuffle
Telomere RNA allows the addition of multiple TTAGGG repeasts to the 3'OH at each telomere
65
What is the telomere shuffle?
3 nucleotides synthesised
Moves forward 6 nucleotides
6 nucelotides synthesised
moves forward 6 nucleotides
66
What is the difference between gel electrophoresis and filtration?
Filtration= (size-exclusion chromatography). Gel is in the form of beads therefore larger molecules move freely between the beads and escape first
Electrophoresis= Continuous mesh of the 2 larger molecules are retarded more
67
Principles of protein interactions
No chemical bonds formed just multiple weak interactions
Surface of these molecules are a poor match and only capable of making a few weak bonds
Thermal motion rapidly breaks apart
Match well so form weak bonds to withstand theremal joining- Ionic, hydrophobic and electostatic
68
What are protein- DNA binding domains?
Several DNA binding domains exist with overall +ve charge to mediate the interaction with the acidic -ve DNA strand mostly through interactions with major groove
69
Several classes of DNA binding proteins containing:
- Leucine zipper motifs- 2 helical structures
- Zinc finger- 2 beta strand with alpha and beta
- Basic helix loop helix (bHLH)- 2 beta strands
- Beta strand
70
What are leucine zipper domains?
Dimers of short coiled-coil sequence and a specific DNA recognition helix
Bind DNA like a clothes peg on a washing line
71
What are Zinc finger proteins?
Recognise specific DNA sequences
metal bound to proteins in many contexts:
-structural eg.Zn
- Regulatory- calmodium
-Catelytic eg Zinc, Iron and copper
Zinc atom domain only big enough to bind a few domains
helical zinc finger rests in groove of DNA
Zn coordinated tetrahedrally
72
Purifying DNA
Binding proteins by affinity chromatography
Silica bead and specific DNA/ oligonucleotide binding -
Total protein get unbound protein
medium to high salt you get DNA binding proteins
73
What is used for gel mobility shift after purification?
Gel elctrophoresis
Salt gradient shows strength of interactions, loading to high salt from medium you can see where they interact- Increase interaction
74
How do we confirm DNA binding?
DNA foot printing (where no cleavage is observed)
DNA radioactive end labelled
- One set added DNA binding protein, DNAse 1 digestion, Remove protein perform gel electroresis auto radiography
-Other set dont add binding protein, DNAse 1 digestion, add to DNA gel electrophoresis
DNAse 1= cleavage agent that cuts at random locations in a sequence manner
75
Results of DNA footprinting
Footprint= where no cleave is observed
| Region of DNA protected by DNA binding protein- cleavage by nuclease or chemical - No footprint as cleavage didn't occur
76
commonly occurring domains
SH2
SH3
PH
77
Insulin signalling pathway involves multiple domain interactions
1. Activated receptor phosphorylates itself on tyrosine and the phosphotyrosine then recruits protein insulin receptor substrate (IRSI). IRSI binds to phosphorinositides on plasma mebrane
2. Activated receptor phosphorylates IRSI on tryrosine and 1 phosphotyrosine binds to the SH2 domain of adaptor protein GRB2
3. Grb2 uses 2 SH3 domains to bind the protein rich protein Sos (binds in phosphotyrosine in the plasma membrane)
4. Grb2 uses 1 SH4 domain to bind in a scaffold (relay signal further)
78
What is the SH2 domain?
Src Homology 2 domain
Phospho lyrosine binding domain
- found originally in Protein lyrosine kinase (SRC) but many other adaptor proteins
- Binding of SH2 is important in signalling complexes
- specificity is between the phosphate of the p-try mainly ionic interactions between -ve and +ve aa
79
What is the SH3 domain?
Uses aromatic amino acid stacking to bind to ligand
Src homology 3 domain
Poly proline binding domain
- SH3 domain involved in linking signalling components and have structural roles in maintaining multiprotein complexes
- Minimum consensus sequence for SH3 binding is p-x-x-p
- contains several aromatic residues- interdigitate between the prolines of pxxp and get trapped by aromatic stacking
80
What is the PH domain?
Pleckstrim homology
- Involved in membrane binding, functions in signalling and anchoring proteins to membrane
- Interact with charged head groups of phosolipids anchoring the protein to domain
- Kinases and phospholipases
81
How do we study protein-protein interactions?
Biochemically- Centrifugation, chromatography, pulldown reactions molecular biology makes it easy to add convient tags to protein
Structurally- NMR, x-ray crytsallography, electron microscopy
82
What tags are used for chromatography or immunopreciptation?
Tag protein of interest with flag peptide DYKDDK
1. Protein of interest
2/ Insert DNA encoding peptide
3. Introduce into cells
4. rapid purification of tagging protein and any associated proteins
83
Immuno pull down of interacting proteins
Mixed tagged proteins with cell extract or other proteins to allow binding of complexes
Add antibody to tag- specific to epitope tag attached to the protein of interest
Add protein A coated beads and centrifuge to recover complex WASH beads.
Identify with mass spec
84
What are common tags for affinity chromatography or pull down?
GST
| Hexa-histidine (6 x His)
85
Common tags for immunoprecipitaion?
HA peptide
Myc peptide
Flag peptide
86
Gsk affinity pull down?
Use fusion protein to pull an interacting protein from a mixture
results in mixture separated by SDS-PAGE and western blotting with antibodies for binding partners
If binding partners unknown use mass spec
87
What are genetic approaches to identifying sequences~?
Yeast 2 hybrid sequence
Colony survival depends on protein interactions- bait protein cDNA (Bait and prey come together and recombinant genes introduced into yeast cell)
Cloned into bait plasmid in host yeast strain (DNA prey proteins)
-interactions between bait and prey after intro, allow DNA binding to activate a reporter gene
-yeast only survive on restrictive growth media
88
How do we measure the strength of protein interactions?
1. Elisa= Enzyme liunked immunogorbent assay
2. Radioactive binding assay
3. surface plasmon resonance
4. Genetic methods of identifying protein interactions
5. live imaging
89
When can protein interactions be studied quantitavely?
When both proteins have been purified
| When you are able to separate the forming complexes from unbound material (SDS, western blot and ELISA)
90
What is FRET?
Fluorescence resonance energy transfer- relies on paired fluorescence
Measures protein interaction, shine violet light and collect green
- violet - protein x - blue
- Blue - protein y - green
91
Radioactivity- based binding experiment
Graph shows amount of A versus bound- binding curve
Curve you can derive the max binding stoichiometry= How many A bind to B and the association constant i.e how tightly A binds to B, which is equivalent to affinity
92
Surface plasmon resosance
The binding of prey to bait increases refractive index of the surface layer
Alters the resonance angle for plasmon induction, which can be measured by a detector
The Kon and K off of a given protein- protein interaction can be read directly from traces
Prey added- association
Buffer wash added-dissociation
93
Measuring protein interactions
At Equilibrium
Association rate = dissociation rate
Kon [A] [B] = K off [AB]
[AB] / [A] [B] = kon/koff=K=Equilibrium constant
94
Visualising macro-molecular protein interactions
Vesicle movement along microtubules relies on kinesin interaction with microtubules and can be studied by light microscopy
Requires:
Kinesin- moves organelles toward and end of microtubules
95
Whtat is the cell cycle?
DNA replication, mitosis, cytokinesis
96
What is the difference between mitosis and meiosis?
Meiosis is is like mitosis but includes amn extra step which creates haploid cells
mitosis each chromosome pair is duplicated to give rise to a sister chromatid
97
Cell proliferation steps
1. chromosome replication and cell growth
2. chromosome segregation
3. cell division
98
Cell cycle has 4 stages
G1- gap (can be very long and permanent)
S- DNA replication
G2
M-mitosis + cytokinesis
99
What is M phase
Prophase (condensation of sister chromatids)- Premetaphase- metaphase (attachment of mitotic spindle to the kinetochore by microtubules) - anaphase (separation of sister chromatids by spindle)- telophase
100
Yeast as a genetic model for cell division
1. Fission yeast - start in G1
2. Budding yeast- Start at end of G1, don't reach homeostasis actively divided
Advantages
- Rapid division 1hr
- Cell cycle control genes are highly conserved
- Yeast can be grown as haploids or diploids
101
How can we study genes that are crucial for cell survival?
Genetic tricks can be used to allow identication of potentiallly lethal mutations:
- Diploids can be used to maintain lethal mutations that are then studies as haploids
- temperature sensitive mutations allow growth at permissive temp
permissive= low temp, goes to M
Restrictive= high temp, restricted at G1
called cell-division cycle genes (cdc genes)
102
Xenopus laevis in mitosis
Oocyte grows- fertilisation - fertilised egg divides without growing
Advantages
- easy to collect eggs
- Rapid division rate
- large size can make purification of proteins easier
- can be maipulated by injection of RNAs or chemical into oocyte
103
What is 'cell-free' mitosis?
Oocyte, sperm and ATP
- one can deplete the cytoplasm of different proteiins using antibodies
- One can remove cytoplasm at different stages to study changes
(eg. In protein phosphorylation)
104
Where are the checkpoints?
1. Start checkpoint- is environment favourable?
- enter cell cycle
2. Enter mitosis- G2/M checkpoint
- Is all DNA replicated?
- I s environment favourable
3. Trigger anaphase and proceed to cytokineses (between metaphase and anaphase)
- Are all chromosomes attached to spindle?
105
What are cyclinally activated proteins?
Cyclins are proteins expressed at different levels during the cell cycle
When present, cyclins bind to specific kinases (Cdk) to activate them
Cdks phosphorylate many proteins that are specific to certain stages of the cell cycle
106
What other proteins modify cdk activity through phosphorylation and binding?
```
P27
cyclins
wee1/cdc25
p27
etc
All send to the next stage of mitosis
```
Active cyclin-cdk complex joins p27- inactive P27-cyclin-cdk complex
107
What is the AP/C checkpoint between anaphase and metaphase
Anaphase-promoting complex- ubiquitin ligase
All cyclins drop at this
-Ubiquintatic E1 and E2 enzymes added to cdk and cyclin
- forms a polyubiqutin chain- degradation of m cyclin in proteasome
108
How is proteolysis controlled by AP/C?
Activating subunit cdc20 and inactive AP/C
109
What is meiosis?
Diploid organisms have 2 versions of each chromosomes (homologues)
Homologues are either maternal or paternal
Only one homologue for each chromosome is packaged into a gamete
Meiosis resembles mitosis but has extra stepthat segregate homologous chromosomes
Pairing of homologues before segregation allows for crossing over (homologous recombination)
110
What are the two stages of meiosis
1. crossing over and segregation
- DNA replication
- Pairing of duplicated
- Line up spindle- crossing over takes place when homologues pair up
- separation of homologs
2. resembles mitosis
- separation of sister chromatids at anaphase 2
- Main difference is that cells in meiosis 2 are haploid instead of diploid
111
What is meiotic prophase 1?
Where homologous pair up
- Pairing is faciliated by the synaptonemal complex as well as DNA base pairing between homologues.
- Homologous recombination between non-sister chromatids serves 2 purposes
1. Aligns chromosomes up read for anaphase +facilitates synaptomeal complex
2. It allows for genetic recombination between paternal and maternal DNA on the same chromosome
112
What happens when there is genetic variation
Mistakes during meiosis 1 result in gamete with an extra chromosome or lacking a homologue- nondisjunction
4% of mammalian sperm is aneuploid and 20% of mammalian eggs
Could cause miscarriage or down syndrome
