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.