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Flashcards in X ray crystallography Deck (49):

what is the fundamental basis of all life?

- chemical interactions


whats the difference between in the old days and now with DNA research?

- in the old days we could only work with abundant proteins, ligands and substrates ( things easy to obtain)
(e.g. hen egg white - lysozyme )
-today - recombinant DNA technologies are used - genes encoding the protein of interest can be cloned and inserted into a plasmid ! ( enhancing protein expression)
- today we only need a very small amount of the material for DNA technology !!
- today we can isolate, purify , exploit PCR etc, we can select and work on exactly what we need.


what are the most commonly used expression system?

- human embryonic kidney cells
( anything that can be cultured in large quantities)


How are cells produced for research?

- these cells are grown in defined media
then labelled ( for NMR) or chemically modified ( for XRAY phasing) amino acids can be added into the protein of interest


What are the steps included in getting more cells of interest...?

1- the expression cells are cultured
2- the protein expression is then induced
3- the cells are harvested by centrifugation
4- broken open and the desired material is removed for the next stage which is


What does the process of Purification involve?

1 - plasmids can introduce a tage onto the protein product ( either N or C terminus )
2-this can be used to form a tagged or fusion protein - (e.g. histidine Tag or a glutathione S- transferase fusion )
3. a tagged protein ( eg histidine tagged protein ) can be passed over a metal ion chelating column (Ni2+)
4. the protein then sticks to the column - everything else passes through
5. the protein target is then eluted using an imidazole wash ( which can achieve a 95% purity from this single column )
N.B other chromatographic methods can be used to puritfy this further - the product can be cleaved to remove the tag and to leave the protein target ( this can be done if a protease cleavage site has been engineered into the expression system --- it isused to cleave Histidinde tag so no fragments are left on the metal ions )


What are the steps in preparing an E.coli Recombinant expression system?

1.the antibiotics resistance gene is in the vector
2- restriction enzyme used to cut at multiple cloning sites on the vector
3. the gene of interest is placed into the vector ( with the antibiotics resistance gene !) ( DNA Ligase used )
4. expression of plasmid
5,. E.coli and plasmid mixed together - under good conditions the E.coli will take up that gene.
6,the E.coli cells are then plated and then further innoculted
7. sometimes you get the gene of interest in the plasmid , or other times the plasmid reseals without it.
N.B. if the expression vector carries a system to allow for colour change - then we see whether the plasmid does what we want - e.g. production of a bluc colour - when B- galactosidase is present OR a white colour - when the enzyme is absent - this allows for selection !


What is IPTG ?

- isopropyl B-D-1 Thiogalactopyranoside )
- used to switch on transcription of the lac operon, which induces the expression of any gene under the control of the lac operator.
IPTG - binds to the lac represser and so switches off repression , rather than switches on transcription.
( it turns off 'molecules' which prevent genes turning on !! )


Once the gene of interest has been found , how can more be made?

- cloned from template or cDNA by PCR
( mutants can be made again using PCR or DNA can be synthesized ready for use!)


Why might we not use E.coli as a system ?

- post translational modifications !


Explain Purification in terms of speed and efficiency?

1. recombinant Protein has been overproduced
2. cells are broken open by mechanical (e.g. sonicator , french press- large amount of pressure)
3. high speed centrifugaation and use of nucleases, filtration can be combined to remove cell debris. -( cell gunk !- reducing viscosity - e.g. E.coli when broken down is like a big viscous soup!!!)
4. if secreted the media is concentrated and the protein may be present at a high level of purity !
5. first chromatography step - affinity column
6. could use a column with antibody - ( a lectin to breakdown the glucose ) - for glycoproteins , transistion metal ions (Ni 2+ )


What is Chromatography?

- Chromotography:- separation between a mobile and a stationary phase
( the sample in buffer= mobile phase , the stationary phase = solid media, spherical particles packed in column )
the stationary phase allows for differential separation by chemical attraction , based on a specific interaction , charge , hydrophobicity or size.
( molecules with small size/charge can be separated as well !!)


What are four methods of Chromatography - and what are their physical/chemical property and resolution?

1.Ion exchange adsorption - charge - high
2.size exclusion( gel filtration ) - size/shape - low
- works from high to low-
3.Hydrophobic interactions -surface hydrophobicity - medium
4. Affinity Chromatography - (Bio) Chemical Binding - high


Describe the basic schematic setup of purification ?

( refer to diagram in lecture 1)


What is Affinity Chromatography ?

-method of separating biochemical mixtures based on a highly specific interaction such (e.g.antigen and antibody, enzyme and substrate, or receptor and ligand.)
- one kind of molecule in the mixture is attached to the molecule that is linked on the resin , other molecules washes through
Two common affinity tags :
- Histidine Tag
( agarose beas linked to NTA ( nitrilotriacetic acid))
- Glutathione S. Transferase fusion ( GST tag)
( agarose bead linked to glutathione binds to this tag)


What is Ion Exchange Chromatography ?

the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
the net surface charge of a protein varies according to pH
pH> isoelectric point (PI) = protein +ve ( anion exchanger )
pH< isoelectric point (PI) = protein -ve ( cation exchanger )
this method involves adsorption to the charged solid resin /support followed by elution in a buffer of higher ionic strength.
( concentrated salt solution can be used to wash the protein away from the medium - charge can be used to separate also.)


What is Hydrophobic interaction ( chromatography )?

- it is a harsh method- can cause trouble with many samples ( was used a lot more commonly in the olden days, affinity chromatography much more common now)
- HIC is based on the reversible interaction between a protein and the hydrophobic ligand bound to the chromatography matrix.
- a matrix
- it depends on how hydrophobic your protein is.
a matrix containing hydrophobic groups binds proteins from aqueous solutions to different extents - depending on the protein structure and a range of other factors ( e.g. salt concentration , pH , temperature)


What is size exclusion Chromatography ?

molecules in solution are separated by their size, and in some cases molecular weight.- usually applied to large molecules such as proteins .
- gel filtration or molecular seive chromatography
- used to investigate protein to protein interactions
partition chromatography to separate molecules of different sizes
porous gel mix - in the form of beads - are used!
- smaller molecules enter matrix pores and move more slowly through the column
molecules of intermediate size can enter the stationary phase - travelling quickly through
- with the correct use of a calibrated column then we can elucidate the mass of the protein = hence its Quaternary structure in solution
gel filtration - larger molecules goes straight to bottom
smaller take longest time.


what methods do we do to assess the level of purity ?

-enzyme or binding assay for activity
- SDS PAGE and Mass Spectometry
- Bradford assay or UV/VIS Spectroscopy for concentration.


What is electrophoresis?

- SDS PAGE ( sodium dodecylsulfate polyacrylamide gel electrophoresis )
-the gel can seperate the proteins on the basis of mass.
- SDS is a detergent and gives uniform charge to the sample. ( the proteins migrate through the gel in the presence of an electrical current)
smaller molecules= move at a faster rate
larger molecules = move at a slower rate
coomassie dye can be used to stain the proteins in the gel and indicate what is present.
(N.B. its the opposite of size exclusion chromatography )


What is the Bradford Assay?

-used to measure the concentration of protein in a solution
- this is important for quantification of interactions.

- N.B. the assay can be influenced by the presence of detergents !

( in testtubes )


What do you know about Coomassie Blue in a Bradford assay ?

- its hydrophobic and charged
-protein binding dye
- under acidic conditions - dye is brown ( adsorption max 465nm )
- in the presence of a protein it is blue ( max wavelength 595nm ) - absorbing red and admitting blue !


Protein concentration :- a spectroscopic method

- aromatic residues absorb UV light at 280nm
- depending on the amino acid sequence we can estimate the molar extinction coefficient for a protein and apply the
Beer-Lambert law

an estimate of (E) - molar extinction coefficient - is taken - but this can affect precision and accuracy of the result.


What is the beer lambert law?

absorption of light - depending on the materials it is passing through


give an equation to understand the way samples are purified?

absorbance coeffient / extinction coefficient


What is Blue Native PAGE?

- a type of gel electrophoresis
- it is when coomassie blue binds target the protein - dye complex - and becomes overall negative
PAGE- under non - denaturing conditions can give information on quaternary structure.


What is gel filtration used for ?

- to investigate Quartnary structures of proteins as wel as protein to protein complexes.


give an example of two proteins which do not / or refuses to interact properly in the gel ?

- Siah 1

normally these two proteins interact , but within a gel thy DO NOT !
they elute on separate peaks - even though they are run through same size exclusion gel , the collected fractions were analysed on SDS PAGE gel!


give an example of two proteins that do interact together in a gel ?

- Siah 1

these two proteins when run on same size exclusion gel - it was seen that the volume is decreased if either protein is run individually. ( this suggests that a Siah 1 - SIP complex has occurred.


what is a summary of sample acquisition?

1. plan experimental approach
2. use recombinant DNA methods and cell growth techniques
3. purify - using HIS or GST atgs
4. use affinity steps - then cleave off tag
5. determine the level of purity


What we need in order to have crystals?

-before diffraction methods can occur - ordered crystals are required to determine a structure

- a pure and homologous sample of protein/ nucleic acids is required for crystal growth

- at least 2-5mg of sample !

- DNA and RNA fragments can be synthesized in quantities and purified by chromatography

N.B. only a few proteins and Ribosomes can be purified for a crystallographic study !


What is a crystal ?

- a crystal is an ordered precipitate , an ordered array of atoms or molecules


what does the process of crystallisation require?

- the process involves achieving supersaturation , the formation of crystal or precipitate nuclei which grows until the system reaches equilibrium.

if non-equilibrium can be maintained then growth can continue.

requires supersaturation and an induction of the crystal nucleus , and a period of time under non- equilibrium conditions.

samples normally between 0.5 and 0.05mm in size !


what is the crystal nucelus?

- the crystal nucleus is a molecular aggregate able to enlist other molecules to join on.


What is nucleation?

- the point where molecules come together and start to precipitate.


what is the supersaturation zone ?

- where more of the sample is in solution than can be dissolved under normal circumstances ( heating can promote this , pH can have profound effect regarding protein solubility )


what is the metastable zone?

- unstable but long lived state, equates to crystal growth.


What is needed for crystal growth ?

we need to take a solution of the molecule of interest and change the conditions to drive to the precipitation zone , achieve nucleation and as the molecules come out of solution they aggregate in an ordered fashion.


How are small molecules ( macromolecules ) crystallized?

- by chance !!!
cooling , slow evaporation , sublimation or vapor diffusion

macromolecule crystals are fragile ! - often unstable and have to be treated carefully and used and fresh


how can proteins and nucleic acids be crystallized ?

- in prinicple they are crystallised in the same way as small molecules ( by chance !!!cooling , slow evaporation , sublimation or vapor diffusion) ,
since we are using the same chemical forces to create and stabilise a crystal lattice
However the physical and chemical properties of biological macromolecules introduces some complications.

- these samples are of greater complexity!
more flexible and sensitive to their environment!
- they must be kept hydrates , at near physiological pH temperature

gentle methods of achieving supersaturation must be employed.


Four main methods for macromolecular crystallisation?

1. Batch method
2. Dialysis
3. Liquid - liquid diffusion
4. vapour diffusion ( the most used method )


What is the batch method?

- mix solution with some precipitant (e.g. NaCl ) until the solution starts to become cloudy , add a little water and the mixture left


What is the Dialysis method?

- sample is contained in dialysis tubing / cassette - pourous membrane which lets solution with some precipitant small molecules through - placed in precipitant solution or a dessicant


What is the Liquid-Liquid diffusion?

- sample is placed in a capillary and , without mixing , a precipitant solution is placed in contact with it. Interface diffusion occurs and promotes precipitation.


What is vapour diffusion?

- most used method!!
- a drop of sample is placed in sealed chamber with a precipitant (e.g.PEG)
- then a precipitate is added to the drop (e.g. protein )
- the relative PEG and macromolecule concentration increases until precipitation occurs


What are the factors affecting Crystal Growth?

- pH
- purity
- ionic strength
- concentration of sample
- type and concentration of precipitant
- colume of sample
- dust
- contaminants
- additives (e.g. metal ion detergents )
- sequence of proteins
- rate of equilibrium

since there are many variables


is radiation good for crystals ?

NOOOO ! as crystals are susceptible to radiation damage , so we protect them with cryo protect (e.g. polyetheylene glycol ( PEG)- whcih cools them down - and then we expose them to X-ray beams !!

( refer to slide 43 on lecture one for diagram )


What are some biophysical methods to investigate affinity , kinetics and thermodynamics ?

- Isothermal titration calorimetry ( ITC)
- fluorescence Thermal Shift Assay (FTSA) -- A.K.A differential scanning fluorimetry (DSF)
- surface plasmon resonance (SPR)
- Bio-layer - interferometry (BLI)


What is ITC ?

- Isothermal titration caloritmetry

meaning of the actual words:

isothermal = constant temperature ( same heat)

titration = small addition of a known concentration of a reagent to a known volume of a molecule of interest ( carefully measured amounts until a reaction or interaction is completed ( seen through colour change , conductance )

calorimetry = measurment of the amount of heat evolved or absorbed in a chemical reaction/ interaction or change of state.

- addition of ligand to protein in the sample cell produces a heat change
- power is increased
- label free technique ( molecule is purified whole time )
- used to characterize the thermodynamics of biomolecular binding interactions by looking at the measurement of heat changes
- it can measure volume of water produced from melting ice over time due to heat / metabolism of rodent

- measures rodent metabolism - as the heat of rodent melted ice and amount of water produced could help determine metabolic rate!!