College 1 - Xray and NMR

Principal of protein crystallography undergoes the following steps:1. Heterologous expression of protein in E. coli.2. Purification of the protein from E. coli.3. Analysis of purity through means of SDS-PAGE.3. Protein crystallization, which is possible through two different methods:

• Hanging drop method• Sitting drop method

4. Screening for protein crystal.5. Optimization of protein crystal.6. Fishing and cryoprotection of crystal. Cryoprotection prevents formation of internal ice crystals, which could potentially damage the protein crystal.7. Data collection X-ray beams hit electrons of protein crystal and reflect, which is dependent on the orientation of electronic distribution.8. Data processing Acquisition of electron density map, which is de skeleton of the protein structure, by Fourier Transforming diffraction patterns.9. Final model is determined through combination of electron density overlap and MM energies.

When you screen for protein crystals, there are three possible phases the protein can be in:- Metastable- Nucleation This is the phase you want your crystal to be in.- Precipitation

When you have found a protein crystal, you want to optimize it, which can be done by changing three different conditions:- Salt concentration- Precipitant concentration- pH

College 2 – Peptides and Proteins

Primary structure Amino acid sequence.Secondary structure -helices, -sheets and turns.α βTertiary structure Protein domain consistent out of secondary structure.Quaternary structure Complex structural assemblages, formed out of tertiary

structures that interact with each other.

Immunoglobulin domain - Consists out of two face-to-face -sheets.β - Every sheet consists out of 3 to 4 -strands.β - Essential for antibody structure.

Zinc-finger domain - 25 aa long and consists of a -helix and a peptide loop.α - Recognizes a specific DNA sequence.

RNA recognition motif (RRM) Recognizes a specific RNA motif.

Important small molecules:- Rapamycin- FK-506

Immunosuppresion through rapamycin undergoes the following steps:1. Rapamycin binds to both FKBP12 and FRAP-FRB/mTOR.2. mTOR is inhibited.3. mTOR can no longer phosphorylate S6K and 4EBP1.4. Translation for genes that activate T-cells is inhibited.5. Immune system is thus suppressed.

Calcineurin pathway undergoes the following steps:1. Calcineurin is activated when bound to CaM.2. Calcineurin dephosphorylates NFAT.3. NFAT migrates into the nucleus.4. NFAT actives genes of immune response.

Immunosuppresion through FK506 undergoes the following steps:1. FK506 binds to FKBP12.2. FK506-FKBP12 complex inhibits calcineurin.3. Calcineurin is no longer able to dephosphorylate NFAT.4. Suprresion of immune response.

College 3 – Chemical Control of Signal Transduction I

There are seven different type of signal transduction pathways:1. Nuclear receptors Steroids, retinoic acid, thyroxine.2. Two-component pathways TGF- , interleukins, interferons.β3. Receptor tyrosine kinases Growth factors.4. Trimeric death receptors TNF- , FasLα5. G-protein-coupled receptors Neurotransmitters, hormones, odorants, tastes,

photons, enzymes.6. Ion channel receptors Glutamate, Na+

7. Diffusible gas receptors O2, NO

Two small molecules for chemical induced dimerization (CID): - Rapamycin- Fusicoccin

Chemical induced dimerization through rapamycin:1. Protein of interest is fused to FKBP.2. Rapamycin binds to FKBP-POI complex.3. Rapamycin induces a strong affinity to bind to FRB.4. FRB is located on plasma membrane.5. Translocation of POI to plasma membrane occurs.

Chemical induced dimerization through fusicoccin:1. Fusicoccin binds to 14-3-3 and PMA.2. Dimerization occurs.

“Anchor-Away” technique Used to change subcellular localization of protein.

Done through CID with rapamycin.

Two types of CID through phytohormones:- Abscisic Acid (ABA)- Gibberellic Acid (GA)

Chemical induced dimerization through ABA:1. ABA binds to ABA receptor.2. Gating loop of ABA receptor closes.3. ABA-ABA receptor complex binds to ABI, a transcriptional regulator.

Chemical induced dimerization through GA:1. Esterase converts GA3-AM to GA3.1. GA binds to GID1.2. N-terminal extension switch of GID1 covers active site.3. GID1-GA complex binds to a GAI or DELLA proteins.

College 4 – Chemical synthesis of peptides

Self-splicing proteins Proteins that have the ability to splice a part of itself from the whole.

The intein of the self-splicing proteins will remove itself.

The two exteins remain and bind to eachother.

The self-splicing method can be used for peptide synthesis:

College 5 – Protein Engineering

Proces om monoklonale antilichamen te produceren bestaat uit de volgende stappen:1. Injecteer muis/rat met eiwit waar tegen je een antilichaam wilt maken.2. Muis/rat maakt antilichaam tegen het eiwit in zijn B lymfocyten.3. Isoleren van B lymfocyten (Blijven niet lang in leven )4. De geisoleerde B lymfocyten worden gefuseerd met een tumorlijn van B lymfocyten waardoor heterocaryonen worden gevormd.5. Heterocaryon is een cel dat beide kernen heeft. De kernen kunnen dan versmelten waardoor een hybridoma cellijn ontstaat.6. Selecteren op hybdridoma cellen die het gewenste anti-lichaam produceren.7. Alleen hybdridoma cellen blijven over, die het gewenste antilichaam produceren en in leven kunnen worden gehouden in een kweek.

College 6 – Chemical Control of Signal Transduction II

Adenylyl cyclase - Protein consistent out of multiple subunits. - Converts ATP to cAMP. - Integrates signaling from different GPCRs.

Forskolin A small molecule that binds the subunits of adenylyl cyclase.

This increases the cAMP levels, which in turn increases activated PKA.

Higher amount of activated PKA results in higher amount of activated CREB, a transcription factor, and thus increase of target genes of PKA.

Brefeldin A (BFA) A small molecule that binds to Arf1 and GEF.

Interaction between Arf1 and GEF is stabilized through brefeldin A, so GEF can’t dissociate as it normally would.

This leads to inhibition of Arf 1.

Inhibition of Arf 1 stops vesicle transport in the cell, because the Golgi will split apart.

* Brefeldin A is a very useful molecule, since it can be used to stop transport in a cell and then analyze a certain compound.

Inflammatory response through ligand TNF- undergoes the following step:α1. TNF- forms a trimer and binds to trimeric death receptor.α2. IKK complex gets activated.3. NF- B is bound to I- B, which is inhibits NF- B.κ κ κ4. IKK complex phosphorylates I- B.κ5. I- B gets a polyubiquitin tail and then a proteasome degrades it.κ6. NF- B gets released and enters the nucleus.κ7. NF- B binds to a regulatory sequence and immune genes are activated.κ

TNF- is an important drug target in immunology, because it starts the pathway for α immune response of the body

Inhibition of this TNF- can be very important for drugs against autoimmune α diseases, since inhibition of TNF- results in suppression of NF- B target genes.α κ

There are two ways to inhibit TNF- :α - Therapeutic antibodies - Small molecules

Immune suppression through means of therapeutic antibodies:1. Antibodies, specific for TNF- , bind to this ligand.α2. TNF- can no longer bind to trimeric death receptors.α3. Pathway for immune response genes can’t activate.

Therapeutic antibody that inhibit TNF- :α- Etanercpt- Infliximab

Immune suppression through means of small molecules:

1. Two TNF- molecules form a dimer. α2. Small molecule binds to this dimer.3. This complex inhibits trimer formation.4. TNF- can only bind to trimeric death receptors in trimer form, so pathway for α immune response genes can’t active.

* This mechanism follows a pre-dissociation-independent binding model.

College 7 – Androgen Receptor Action

Androgen receptor Receptors for sex hormones, such as testosterone.

The pathway for gene activation through androgen receptors undergoes the following steps:1. A single AR is bound to a HSP.2. DHI binds to a single AR.3. AR dissociates from HSP.4. AR dimerizes and enters the nucleus.

5. Dimer binds to a specific sequence and gene transcription is activated.

The natural agonist binds to the binding pocket of the androgen receptor through hydrophobic interactions.

Inhibition can be accomplished through an antagonist, which is slightly larger than the agonist itself. This means that the agonist itself can no longer enter the binding pocket, because of steric strain.

The antagonist can be used as an anti-cancer drug for tumors in sex organs.

However, this does come with two possible problems, because tumors mutate very quickly:

1. Mutations of tumor cells, that enables production of AR/ER in which antagonists can’t bind.2. Mutations of AR/ER in such a way that the antagonist becomes an agonist and even increases AR activity.

A key amino acid mutation and it’s consequence:W741 is mutated to L741. Tryptophan is a larger amino acid than leucine, which means the binding pocket becomes bigger and the antagonist can now act as an agonist and no longer can work as an anti-cancer drug.

College 8 – Microfluidics for Chemical Biology

Applications for microfluidics:- Drug testing- Protein evolution- Peptide synthesis- Protein crystallization

A microfluidic device out of PDMS can be made with two methods following each other consecutively:

- Photo lithography Making a mold on a silicon wafer using UV light to etch a design.- Soft lithography Using the mold to make a chip from PDMS polymer

In photo lithography you fabricate a master for your microfluidic device, which is done in the following steps:1. Spin-coat photoresist on a silicon wafer.2. Place a photomask, which has the pattern for your microfluidic, on the photoresist.3. Expose photoresist to UV light through the photomask.4. Develop exposed wafer with photoresist, during which the parts of the photoresist that were not exposed to UV light dissolve.

In soft lithography you replicate the master mold in PDMS, which is done in the following steps:1. Pour PDMS monomer and cross-inker mixture onto the master mold.2. Cure and peel-off PDMS.3. Cut out the replica.4. Create access ports.5. Bond the PDMS replica to a glass slide.

College 9 – Enzymes, Kinases, Proteases, Activity-based profiling

The human body knows a vast amount of different kinases.

There are inhibitors for these kinases, but most are general kinase-inhibitors and not specific enough if you want to evaluate the function of a specific kinase.

Kinase-specific inhibition can be achieved by coupling chemistry and genetics, through the following steps:1. There is a general inhibitor for the kinase of interest.2. The general inhibitor is modified, in such a way that it can no longer bind to binding

pocket of kinases. For example making the inhibitor bigger.3. Mutate the binding pocket of the kinase of interest in such a way that the modified inhibitor can bind to the mutant kinase.4. The modified inhibitor now only works in the mutant kinase.

Activity-Based Protein Profiling (ABPP) Protein tagging based on activity.

There are multiple types of ABPP:- Gel-based ABPP- (LC/MS)-based platforms for ABPP- (TOP)-ABPP- Competitive ABPP

ABPP is a very useful proteomic technology, on which bases you can check which enzymes are inhibited and which are not.

Gel-based ABPP is done through the following steps:1. Multiple enzymes/proteases are present and some are inhibited.2. ABPP probe is added and they only bind to active proteases, so none that are inhibited.3. Active proteases, on which an ABPP probe is bound, become fluorescent.4. Through SDS-PAGE it can be checked which proteases are active and which are not.

(LC/MS)-based platforms for ABPP can be used for identification of peptides and their labeling site. ABPP for peptide identification is done through the following steps:1. ABPP is added to bind to an active protease.2. Streptavidin is then added to make the probes visible. 3. Trypsin is added to chop the protease in many peptides.4. Liquid chromatography-mass spectrometry is performed in order to identify peptide of active protease.

ABPP for labeling site identification is done through the following steps:1. ABPP is added to bind to an active protease.2. Trypsin is added to chop up all proteins.3. Streptavidin is added to bind to the probes, which are in turn bound to peptides of labeling sites.4. Liquid chromatography-mass spectrometry is performed in order to identify labeling site.

(TOP)-ABPP is a new technique which can be used for simultaneous characterization of protein targets of probes and sites of probe labeling.

(TOP)-ABPP is done through the following steps:1. Reactive group of a “Clickable” ABPP probes are added to bind to active proteins.2. TEV-tag is added, which binds to the reactive group and together make up the “clickable” ABPP probe.3. Streptavidin is added and binds to the TEV-tag.4. - If labeling site is of interest, trypsine is added and then LC/MS is performed. - If the protein itself is of interest, TEV protease is added and then LC/MS is performed.

College 10 – Fluorophores and Labeling of Proteins

There are four types of protein labeling:- Metal-ligand interaction-based labeling- Biological recognition-based labeling- Self-modifying enzyme tag interaction-based labeling- Enzyme-mediated labeling of specific peptide sequences

Metal-ligand interaction-based labeling is done through the following steps:1. Expression of protein of interest modified with a tetracystine motif. 2. FlAsH-EDT2 binds to the tetracystine motif through disulfide bonds.3. FlAsH-EDT2 becomes fluorescent.

Biological recognition-based labeling is done through the following steps:1. Protein of interest is bound to enzyme eDHFR.2. Small molecule TMP binds to eDHFR.3. TMP becomes fluorescent.

Self-modifying enzyme tag interaction-based labeling is done through the following steps:1. Protein of interest is bound to enzym hAGT.2. hAGT binds covalently to EGAF, and a part of this small molecule dissociates.3. EGAF is a fluorescent molecule, so the protein of interest is now labeled.

Enzyme-mediated labeling of specific peptide sequences is done through the following steps:1. Protein of interest is bound to the enzyme LAP.2. LpIA and coumarin are added.3. LpIA acts as an peptide acceptor for LAP, and thus they bind together.4. Structure becomes fluorescent.

SNAP-tag hAGT functions as a snap-tag and is fused to a protein of interest.

A fluorescent label can be attached to a O6-benzylguanine derivatives.

Fluorescent label leaves O6-benzylguanine derivatives and binds to hAGT.

Protein of interest now has a fluorescent label and can be tracked in the cell.

CLIP-tag Same principle as in SNAP-tag, but hAGT now can accept O2-benzylcytosine derivatives.

* Through a combination of CLIP-tags and SNAP-tags two fusion proteins can be labeled simultaneously and specifically.

College 11 – Small-molecule Screening, biochemical assays…

Key factors for HTS screening:- Time: • Time/well • Wells/day

• Screens/year • Screens/year • project time- Costs: • Reagents • Consumables • Instrumentation • Personnel- Quality: • Few false positives • Few false negatives • Signal/noise ratio • Validated ‘Hits’ Enzyme-linked immunosorbent assay (ELISA) A screening technique involving

antibodies to identify a substance.

ELISA is performed through the following steps:1. Antigens from a sample are attached to a surface.2. An antibody, which is linked to an enzyme, binds to the antigen.3. Substrate for enzyme is added, which binds to the enzyme.4. This produces a detectable signal.

Fluorescence Polarization assay (FP) Technique to verify protein presence through polarized light.

FP is performed through the following steps:1. A ligand with a fluorescent label is rotating rapidly and light becomes depolarized.2. Target protein is added.3. If protein binds to ligand, the complex will rotate slower and light will remains

polarized.

* If you also add an inhibitor, the ligand will remain unbound. This means the light remains polarized. This is also an effective way to examined if a ligand is functional for a specific receptor protein.

Molecules excite a wavelength of their own color.

This creates false positives in HTS.

TR-FRET Time-resolved measurement of fluorescence resonance energy transfer.

TR-FRET is performed through the following steps:1. Donor molecule is excited and sends out photons.2. If donor molecule and acceptor molecule are close enough, the emission photons excite the acceptor molecule.3. Acceptor molecule emits polarized light.

4. Time delay of 50 to 150 µ seconds between excitations and fluorescence measurement eliminates short-lived background fluorescence.

ALPHA screening Amplified Luminescent Proximity Homogeneous Assay.

ALPHA screening is performed through the following steps:1. Donor bead gets excited through a photon.2. If donor bead and acceptor bead are close enough, the donor bead will donate O2.3. Acceptor bead accepts the O2.4. Acceptor bead sends out photons.

College 12 – DNA: Synthesis, Sequencing, and Therapy

Three different methods for DNA sequencing are:- Capillary electrophoresis- Dideoxy sequencing- Pyrosequencing

Capillary electrophoresis is performed through the following steps:1. Oligonucleotides, bases tagged with fluorescent groups are separated within glass capillary filled with ionic buffer.2. Positively charged cations drag oligonucleotides with them to the negatively charged cathode.3. The electrophoretic movement of oligonucleotides largely depends on size and charge.4. Laser detects fluorescent molecules, and thus can tell what bases are detected.

* Nucleotides may be maximally 500 nucleotides long.* Sensitivity of a single base.

Dideoxy sequencing is performed through the following steps:1. Starting mixture of template strand, short complementary primer, regular dNTPs and magnesium chloride.2. A mixture of small amounts of ddNTPs is added.3. DNA Polymerase starts the polymerization reaction.4. Elongation occurs until a ddNTP is incorporated in the growing strand, because the ddNTP blocks the polymerization.

5. Mixture of fragments, which each end with a distinctive fluorescent nucleotide, are analyzed by capillary electrophoresis.

Pyrosequencing is performed through the following steps:1. DNA fragments are attached to beads, each in a different container.2. A DNA polymerization reaction is performed in each container, by constantly adding one dNTP.3. If dNTP can bind in the container, a pyrophosphate is released.4. Pyrophosphate binds to APS and forms ATP.5. This creates a signal in the form of a light pulse, due to the fact that ATP is a substrate for luciferin.