Post on 28-Aug-2019
transcript
Summary Chemical biology
Index
College 1 structural biology .................................................................................................................................... 3
NMR .......................................................................................................................................................................... 3
X-Ray ......................................................................................................................................................................... 3
College 2 Proteins - I ................................................................................................................................................ 5
Protein function ..................................................................................................................................................... 5
Rapamycin ............................................................................................................................................................... 5
FK506 ........................................................................................................................................................................ 5
College 3 Chemical control of signal transduction – I ..................................................................................... 6
Chemical induced dimerization (CID) .............................................................................................................. 6
Bump-and-hole approach: ................................................................................................................................. 6
College 4 Chemical synthesis of peptides ........................................................................................................... 7
Amino acids: ........................................................................................................................................................... 7
Problems and solutions by solid phase peptide synthesis: ........................................................................ 9
College 5 Chemical control of Signal transduction – II ................................................................................. 10
Pathways: ............................................................................................................................................................... 10
College 6 Enzymes, kinases, proteases, activity-based protein profiling ................................................. 12
Design of inhibitor proteins .............................................................................................................................. 12
Activity-based protein profiling (ABPP) ......................................................................................................... 12
College 7 Protein engineering ............................................................................................................................. 13
Enhancing protein stability ................................................................................................................................ 13
Proteins that resist boiling ................................................................................................................................. 13
Generation of antibody specificity .................................................................................................................. 13
Display methods .................................................................................................................................................. 13
College 8 Protein labelling, high-throughput screening .............................................................................. 15
Protein labeling techniques: ............................................................................................................................. 15
Calcium and zinc sensors .................................................................................................................................. 15
Monitoring protein-protein interactions ....................................................................................................... 16
College 9 Nuclear receptors ................................................................................................................................. 18
College 10 Microfluidics ......................................................................................................................................... 20
Basic principle of microfluidics ......................................................................................................................... 20
Making a microfluidic device ............................................................................................................................ 20
College 11 DNA: synthesis, sequencing and therapy .................................................................................... 21
Automated DNA synthesis ................................................................................................................................ 21
Dideoxy sequencing ........................................................................................................................................... 22
Pyrosequencing ................................................................................................................................................... 22
College 1 structural biology
NMR
How does it work?
Every protein exists of a unique composition of protons, by placing them in a
magnetic field the protons align in the same direction. When the protons receive a
pulse they will flip 90o and send the energy back. The time delay of the energy that
is send back due to difference in proton density can be measured. These
measurements can then be calculated back to a protein structure. This process is
very quick but can only be applied on small molecules.
X-Ray
How does it work?
X-Ray crystallography makes use of protein crystals. In a crystal all proteins are
aligned in exactly the same configuration. If an X-ray beam is applied on the
crystal the electrons of the protein will scatter this beam in a specific way. This
elector scatter can be measured and computed into a protein.
What are the parameters to describe the quality?
How to achieve proper crystals?
Two main way of growing
crystals: hanging drop and
sitting drop. Both work on
the same principle. A drop
made of protein and
reservoir, containing salt
precipitant and buffer, is put
in a space where there is
another reservoir with the
same substances as the drop but in a much higher concentration. By
diffusion/osmoses/precipitation the reservoir of the drop will crystalize.
Only in perfect conditions the crystal will be created. The crystallization can be
under saturated (nothing will happen) or
supersaturated (crystal will be formed). The
supersaturated form knows three phases.
Clear (metastable) > nucleation >
precipitation. Only in nucleation a proper
crystal will be formed.
The conditions of the crystal depend on
three parameters: pH, precipitant and salt
concentration. Only in the right
combination a proper crystal is formed.
What is the function of cryo protection?
The crystals are beamed with x-ray which has very much energy and boils the
proteins which would denature the proteins. Therefore the proteins are frozen with
liquid nitrogen but since the crystals exist of +/- 80% water ice crystals would form
which would negatively affect the proteins. To freeze the crystals but keep the
water from becoming ice the crystal is protected by cryo.
Quality of protein:
The quality of the protein can be described by a couple of things.
The resolution in angstrom (Å).
The R-factor, an error factor that describes the disorder of the whole protein.
Given between 0 and 1
The B-factor describes the apparent disorder of the protein for each atom/amino
acid. When a protein is still moving, there is a high B-factor.
The occupancy rate is the rate of occurrence of a conformation of the protein. To
get a good result the occupancy should be 70% or higher.
College 2 Proteins - I
Protein function
Kd:
Kd value describes the concentration of protein when 50% of is bounded.
𝐾𝑑 =𝐾𝑜𝑓𝑓
𝐾𝑜𝑛
Koff describes the dissociation
𝑅 ∙ 𝐿 → 𝑅 + 𝐿
Kon describes the association
𝑅 ∙ 𝐿 ← 𝑅 + 𝐿
The Kd values of different interactions and different proteins can easily be
compared.
EC50:
EC50 is a similar kind of value as Kd but it is a value that indicates the rate of
biological activity instead of protein interaction. It sets the dose against the
response.
Rapamycin
Rapamycin is a molecular glue that binds FKBP12 to FRAP-FRB/mTOR. mTOR usually
phosphorylates S6 kinase which phosphorylates
S6. mTOR also phosphorylates 4EBP1. Both S6
and 4EBP1 start translation, cell growth and
activation of T-cells.
Rapamycin binds hydrophobic to FKBP12 and
FRAP-FRB. But it also has H-bonds with FKBP12
FK506
FK506 is also an immune suppressor. It is a molecular glue that binds to FKBP12 just like
Rapamycin but instead of binding to FRAP-FRB it binds to the Calcineurin A-B complex.
Calcineurin normaly dephosphates NFAT which if unphosphorylated can start translation
of immune respons genes.
College 3 Chemical control of signal transduction – I
Chemical induced dimerization (CID)
Chemical induced dimerization is a simple way to bring specific proteins together. FRAP-
FRB can be connected to a specific protein or to the cellmembrane or an organell. If
rapamycin glues FKBP12 and FRAP-FRB together FKBP12 will also be connected to the
specific protein. When a protein of interest (POI) is bound to the FKBP12 you can bring the
POI and the other protein together.
Two other CIDs can be made by the use of the phytohormones: Abscisic Acid (ABA) and
Gibberlellic Acid (GA). The dfference between these and rapamycin is that they don’t bind
to two proteins but that they are enclosed by one protein which makes it able fot the
other protein to bind.
The phytohormones do not interact with rapamycin and thus the both CID systems can be
combined.
Fusicoccin is another protein that can induce CID. It can couple a 14-3-3 protein to
another protein (mCherry) even if this protein is not in the cytosol but in the nucleous. The
coupling of 14-3-3 and mCherry can induce the proteation of I-κB which then makes
NF-κB active.
The advantage of Fusicoccin in comparison to rapamycin is that rapamycin has a much
stronger binding affinity to FKBP12 than Fusicoccin has to 14-3-3. Which means that when
you want to stop the dimerization by washing out the Fusicoccin or rapamycin that this
won’t work for rapamycin.
Bump-and-hole approach:
To make sure that rapamycin only binds the FKBP12 that is connected to the POI you
change the bump of the rapamycin and the hole of the FKBP12 so that when you add the
specifically designed rapamycin it will only bind to the FKBP12-POI dimer.
The changes in bump-and-hole can be for instance to change a small amino acid to
tryptophan on the bump and change an amino acid in the hole to a small glycine.
College 4 Chemical synthesis of peptides
Amino acids:
One letter code – structure, properties, pKa and charge at pH:
Name 1 letter
code
structure properties pKa protonation
glycine G
Small -
alanine A
Small -
valine V
Aliphatic -
leucine L
Aliphatic -
isoleucine I
Aliphatic -
proline P
Inflexible -
serine S
H-bonding 13.5
threonine T
H-bonding 13.0
tryptophan W
Aromatic,
bulky
-
phenylalanine F
Aromatic -
tyrosine Y
Aromatic(,
acidic)
10.1 0.13%
deprotonated
cysteine C
Sulfide
bonding
8.3
methionine M
non-polar
histidine H
Basic 6.0 6%
protonated
+
lysine K
Basic 10.8 99.97
protonated
+
arginine R
Basic 12.5 99.9995%
protonated
+
asparagine D
Amine -
glutamine E
Amine -
aspartate N
Acidic 3.9 99.95%
deprotonated
-
glutamate Q
Acidic 5.3 90.0%
deprotonated
-
Problems and solutions by solid phase peptide synthesis:
During SPPS a lot of problems can occur such as: incomplete reactions
(aggregation & secondary structure formation), side reactions can occur such as
cleavage of the resin, accumulation of nearly identical impurities make purification
very difficult and There is a limit of 40-60 amino acids.
There are few solution to these problems.
The resin can be modified in such way that the peptides are thus far from each
other that they hardly
aggregate (tentagel resin).
To prevent the peptides to form secondary structures in an early phase
pseudoprolines can be
introduced to make a nick in
the peptide which ensures that
the peptides won’t align
properly.
Another way is to protect the
nitrogen groups with
Dimethoxybenyl(Dmb) to
prevent structuren from H-
bonding.
To prevent peptides to form S-bonds Native Chemical Ligation (NCL) can be
applied on the peptides. This way the cysteines will be created later in the
synthesis.
College 5 Chemical control of Signal transduction – II
Pathways:
Receptor tyrosine kinase:
Fibroblast growth factor receptor (FGFR)
FGFR is the receptor of the fibroblast growth factor (FGF). It is a receptor tyrosine
kinase that once bound to FGF will self phosphorylate to make a docking stations
and starting points for multiple pathways. Two of those pathways are described:
Phospholipase C γ (PLCγ) pathway:
Once phosphorylated PLCγ can
bind to the RTK. When bound it
can activate PIP2. Activated PIP2
can bind to DAG but it can also
transform into IP3. The IP3 can
release Ca2+ from the ER. Now
the DAG and Ca2+ can induce
transcription.
Proliferation and anti-apoptotic signaling through Akt:
The FGFR can also phosphorylate SHC1 which after a cascade transforms
PIP2 into PIP3. PIP3 can bind PDK1 and Akt to the membrane. When PDK1
and Akt are closely together Akt will be activated. Activated Akt does
multiple things:
It inhibits GSK-3 which normally inhibits mitose/differentiation.
It inhibits TSC•TSC2 which normally inhibits protein translation.
It inhibits BAD which normally activates apoptosis.
It inhibits FOXO1 which normally activates apoptosis.
It activates HDM2 which normally inhibits apoptosis.
G-protein coupled receptor (GPCR):
The PLCγ pathway can also be triggerd by another kind of receptor, GPCR. The α-
part of the GPCR can be
activated by an GTP. After
this the α-part can activate
PLCβ which starts the same
pathway as PLCγ. But now
the Ca2+ ions can also
activate calmodulin which
ensures muscle contraction
and transcription.
Frizzled (β-catenin)
The GPCR Frizzled can be activated by Wnt. When activated, the α-part of Frizzled
will dissociate and
together with Dsh
inhibit the Axin-APC-
GSK-3 complex that
normally phosphor-
ylates β-catenin. If β-
catenin is phosphor-
ylated it will degrade if
not it will bind with Lef-
1 and start mitosis.
Tumor necrosis factor receptor (TNFR)
TNFR is a trimeric death receptor that can induce
inflammation. When bound to the trimer TNFα it
activates NIK which activates IKK. IKK ensures that the
Inhibitor κB protein, that is connected and inhibits
NFκB, will be degraded leaving the NFκB to start
transcription of inflammation genes.
Inhibition of TNFα
TNFα can be inhibited by a small molecule that
can ensure that the trimer will fall apart into a dimer and monomer.
There are two models that describe this inhibition. Model 1 states
that the trimer self-reacts into a dimer and monomer. When that’s
happens the small molecule can react with the dimer so the trimer
can’t be formed. Model 2 states that the small molecule will just sit
in the trimer and dissociates one monomer. Model 2 is most likely
to happen since spontanious dissociation is very unlikley.
College 6 Enzymes, kinases, proteases, activity-based protein
profiling
Design of inhibitor proteins
The trick of the bump-and-hole approach is that you change the kinase and inhibitor so
they will only fit to each other but in such a way that the change won’t affect the natural
working of the protein kinase.
Activity-based protein profiling (ABPP)
To see which proteins in a tissue are active you can connect a Tag to a reactive group
which can bind to active proteins. If you let the reactive group + tag react with the
proteins of a tissue you can
see which proteins are active
and which aren’t by
preforming an SDS-PAGE. If
you do this with healthy and
sick tissue you can compare
them and see which proteins
(might) cause the disease.
If you want to know which
exact proteins they are you
can separate them by
binding the tag to a molecule
and then performing an
LC/MS.
College 7 Protein engineering
Enhancing protein stability
There are multiple ways of enhancing the stability of proteins:
- Introduction of disulfide bonds
- Maximize the hydrophobicity in the protein interior
- Stabilize polar resideus in the protein interior by
Salt bridges
Helix dipole
- Replacement of glycine by proline
Proteins that resist boiling
3 ways of enhancing the resistance of boiling of a protein are:
- Replacing unimportant amino acids by proline
- Salt bridges
- Disulfide bonds
Generation of antibody specificity
The highly specific diversity of antibodies is a result of 3 diversity sources:
- 150 different combination of V, J, D and C genes in the loops of the heavy and
light chain.
- Default pool of antibody producing cell that generate 108 different antibodies?
- Point mutations in the recombined genes by making mistakes on purpose.
Display methods
When you have a library of proteins and want to find out which one has the highest
affinity to your target you
put them in a solution
and add this to your
target. After washing the
unbound protein you will
be left with the protein
which has the highest
affinity. Sadly though not
only the best but also
proteins with semi good
affinity will stay behind.
Cell display
To solve this problem you add the DNA sequence to you’re a cell and let it get to
expression on the cell surface. Now the cell and protein are physically connected
to each other. So after washing you will have a few cells which show different
binding affinity to your target. To determine which has the best affinity you elute
the high affinity cells from the target, perform a PCR and add them to your target
again. Now after washing the protein-cell complex with the highest affinity will
attach most to
your target. If
you repeat this
PCR and target
step a couple
of times you
will end with
the protein-cell
complex with
the highest
binding affinity
to your target.
Phage display
Phage display another display method similar to cell display. Only you can’t
perform PCR on a phage but you have to multiply them by inserting them in E.
Coli.
Ribosomal/mRNA display
A same kind of cell display is Ribosomal display. Now you make an RNA-string of
your protein. A ribosome will make a protein of this. If you would design te protein
in such a way that it won’t depart from the ribosome you can have your RNA-
string physically attached to your protein. Now you can get the protein with the
highest affinity to your target the same way as with cell display.
College 8 Protein labelling, high-throughput screening
Protein labeling techniques:
There are a lot of different ways of binding a tag to a protein or peptide. You can have a covalent
bond which means that the tag is directly linked to a molecule or protein.
It can be noncovalent which means that the molecule (mostly ligand or substrate) on which the
tag is bound interacts with a part of the protein of interest but doesn’t have molecular bonding.
Instead they have S-S bonds or H-bonds etc.
You can also covalent or noncolalent bind a tag to your peptide.
Another way to bind a tag to your peptide is by use of an enzyme.
The sizes of the tags differ very much from 12-33 kDa for proteins to 5-33 amino acids for
peptides. Often the bigger a tag is, the more specific it binds to a protein but if a tag is to big it
can have an influence on the working of the protein.
One way to bind a fluorescent tag to a protein is by inducing a cysteine rich part in you protein
and a sulfate rich part in your tag so they can make disulfide bonds. This is called metal-ligand
interaction-based labeling
Another way of binding a fluorescent tag is biological recognition-based labeling. Similar to
chemical induced dimerization a tag can be bound to a (FKBP12) and your protein of interest can
be bound to another protein (mTOR) which can be ‘glued’ together by rapamycin.
The use of enzymes to bind tags to proteins of interest is also a very much used technique.
SNAP-tags and CLIP-tags are very useful tools of binding a tag to a protein of interest. They make
use of the DNA-repair molecule O6-
alkylguanine-DNA alkyltransferase (hAGT).
SNAP-tags and CLIP-tags are DNA
nucleotides which have an extra benzene and
label attached to them. The hAGT splits the
nucleotide and binds the benzene-tag.
Calcium and zinc sensors
To determine the presence or quantity of Ca2+ and Zn2+ ions a special molecule is
designed. The molecule exists of two fluorescent proteins with different excitation and
emission spectra. They are connected with a linker molecule and a calcium or zinc binding
protein. When Ca2+ or Zn2+ binds the two fluorescent proteins come closer to each other.
If the two proteins are close enough Fluorescence Resonance Energy Transfer will occur.
By excitation light of one of the proteins you can see whether if calcium or zinc is present.
Monitoring protein-protein interactions
There are multiple techniques of monitoring protein-protein interactions. A couple of
these techniques explained here.
The SNAP- and CLIP-tags can be used at the same time but also together, this is called (S-
CROSS). Two different proteins are bound to
SNAP or CLIP, when they are bound together.
Now the SNAP and CLIP are very close and can
bind a fluorescent linker.
Another way of investigating protein-protein interactions is with a protein
complementation assay (PCAs). Here a tag is modified into two parts which together are
fluorescent but separated do nothing. Each of these parts is
linked to a protein thus when the proteins interact the tag
will be fluorescent.
Fluorescence polarization (FP):
Proteins are polar and there for rotate. When they are beamed with polar light the
light will be depolarized. When the protein interacts with another protein the total
mass will be higher and the
rotation will be slowed
down. The depolarization
will be lower. The difference
in depolarization can be
measured and the amount
of protein-protein
interactions can be
determined.
Enzyme-linked immunosorbent assay (ELISAs):
To detect very small quantities of antigens (proteins, peptides, hormones etc.)
ELISAs can be used. ELISAs binds the antigen to a wells plate. After this antibodies
are added. The right antibodies will bind the rest will be washed away. After this
another antibody, which has a tag added to it binds to the first antibody. Now you
can see in which well the antigen is present.
Time Resolved-FRET (TR-FRET)
Works similar like calcium and zinc sensors bud has a measurement window that is
delayed so it won’t measure background fluorescence.
ALPHA screen
Works the same way as FRET but it doesn’t
use FRET but two molecules that can
diffuse oxygen. When the donor bead gets
beamed it diffuses oxygen to the acceptor
bead which emissions light. In a different
color than the donor bead.
College 9 Nuclear receptors
Nuclear Receptors Steroids retinoic
acid, thyroxine
Two component
pathways
interleukins
RTKs EGF
Trimeric death TNFα
G-coupled neurotransmitters
Ion channel Na2+
Diffusible gas
receptor
NO2, O2
Oestradiol bindt aan de estrogen receptor.
Estrogen receptor heeft twee leucines (functioneren als
steric hinderance zodat de estradiol niet in het
verkeerde vlak bindt, een glutamine, een arganine en
een histidine vormen waterstofbruggen met het
oestradiol).
Klasse II Nucleaire Receptoren - stof zorgt voor
ligand dimerisatie in nucleus- genexpressie
(kortom de afbeelding hiernaast)
Steroid hormoon-stof zorgt voor eiwit
dimerisatie en verval in plasma - import nucleus
via kanaal en gentranscriptie (kortom afbeelding
hieronder).
DNA Bindings Domein
- twee helix binden in major groove
- gestabiliseerd door zink vingers, met
omringende cysteines. (Dus de plek
waar eiwitten binden aan DNA)
Binding co regulators op genen voor het uitvoeren van activatie/deactivatie
chromosome remodeling
- histone acetylation= Transcription of chromosome condensation
- histone deacetylation = NO transcription
Antagonist = neemt bindingsplaats in en inhibeert (Tamoxifen = Estrogen antagonist)
Agonist = exacte kopie die zelfde taak vervuld als originele eiwit
Four chemical biology concepts:
1. Using insect NRs to switch on human genes
2. Targeted protein degration
Using an chemical induced dimer to bring a protein to ubiquitin. After
ubiquitination the protein will be degraded.
3. Protein splicing
An intein is a segment of a protein that is able to excise itself and join the
remaining portions (the exteins) with a peptide bond in a process termed protein
splicing. Inteins have also been called "protein introns". Intein-mediated protein
splicing occurs after the intein-
containing mRNA has been translated
into a protein. This precursor protein
contains three segments — an N-
extein followed by the intein followed
by a C-extein. After splicing has taken
place, the resulting protein contains
the N-extein linked to the C-extein;
this splicing product is also termed an
extein.
4. Conformational sensors
As explained in college 9 with calcium and zinc sensors
.
College 10 Microfluidics
Basic principle of microfluidics
Microfluidics are devices in which you can sense, pump, mix, monitor and control fluids on
fl scale. You can make devices which can execute laboratorial processes such as dilution
series etc. The advantages of these devices in comparison to the normal processes are
that it is faster, uses less reagent (and is thus cheaper), can be intergrated with other
devices (electrodes/machnetic fields on a much smaller scale and you can run parallel
measurements so it is faster.
On these small scales the flow of the fluids is laminar which means that you can predict
and describe the flow very precise and easy. Laminar streams do not mix, they only
undergo diffusion with other streams.
Making a microfluidic device
These microfluidic devices are made with use of two types of lithography:
Photolithography and soft lithography. Photolithography is the first step in which you
make a mold of silicon wafer using UV light. In soft lithography the mold is used to make a
chip from polydimethyl siloxane (PDMS) polymer.
Steps to make a microfluidic device:
- First you have to make a mask in software like CAD
- Then you have to print this mask with a laser-writer. This mask has to have a
- layer of photoresist. There are two types of photo resists: positive and
negative. Negative photoresist becomes insoluble after exposure to UV light.
- Photolithography
Next you have to make a very thin wafer
plate by spinning SU-8 at 200 oC.
After this you have to cover the wafer
with your mask and expose it to UV
light. A part of the wafer will be soluble.
Now you have to process the wafer to
remove the soluble SU-8 so you will end
with and master mold of your print.
- Softlithography:
Poor PDMS on the mold to make a polymer and
let it rest for a few hours.
Peel of the polymer from the master mold
- Punch holes for tubes for inlet and outlet of fluid
- Attach the PDMS to a glass plate by an oxygen plasma
treatment
- Insert tubes and fluid pumps
College 11 DNA: synthesis, sequencing and therapy
Automated DNA synthesis
DMT is een beschermende groep voor de vrije OH aan de suiker. De groene circels geven
beschermende groepen
aan voor de vrij NH
groep van de
nucleotide. Cyanoethyl
phosphoramidite wordt
gebruikt als resin.
Hierna kan de automated DNA synthese beginnen.
1. Met TCA de 5’ OH-groep vrij maken, dus DMT eraf halen.
2. Tetrazole en phosphoramidite reageren snel samen. Dit reageert later met de vrije 5’ OH-groep.
3. Capping: op alles wat niet heeft gereageert wordt een cap gezet, zodat deze niet meer verder
zullen reageren in de volgende stappen.
4. De fosforgroep wordt geoxideerd waardoor deze stabieler wordt.
Stappen 1 t/m 4 worden herhaaldelijk uitgevoerd.
5. De beschermende groepen worden eraf gehaald met TCA. CPG (resin) wordt verwijderd.
Dideoxy sequencing
Aan een single streng DNA (gedeeltelijk als wel dubbelstrengs want anders kan DNA
polymerase niet verder gaan, heeft een begin nodig) worden naast de gewone
deoxynucleotide ook fluorescente
dideoxynucleotides (>1%)
toegevoegd. Als deze zijn gebonden
stopt de DNA synthese omdat de vrije
3’ OH groep ontbreekt. Hierna
worden alle DNA strengen gescheiden
door gel elektroforese. De kleinste
sequenties worden het eerste gescand
door de laser. Uiteindelijk kan worden
bepaald wat de totale sequentie was
aangezien elke base op het einde een
andere fluorescente kleur heeft.
Pyrosequencing
Elke keer wordt een overvloed dezelfde fluorescente basen aan een single streng DNA
toegevoegd. Als deze binden aan de DNA keten geeft dit een fluorescent signaal. De
grote van het signaal geeft aan hoeveel basen er gebonden zijn. Na deze stap worden
alle niet gebonden basen weer afgebroken en worden de volgende fluorescente basen
toegevoegd.