Flashcards in Semester 2 Deck (113):
How does gel filtration work?
- molecules in solution separated by size as passed though column packed w/ gel matrix
- matrix consists of porous beads
- smaller molecules can diffuse further into pores of beads
- larger molecules can't enter many pores (if any)
- so largest eluted first and smallest last
- the smaller the molecule, the more of the total vol of column it passes through, so greater the vol of eluent req to elute it
What is the vol of eluent proportional to in gel filtration?
- time taken for collection
What is the result of using small beads in gel filtration matrix?
- less diffusion, so elution peaks sharper and separation better?
What is void volume (Vo)?
- molecules excluded from beads elution vol, equal to liquid outside beads
What is elution volume (Ve)?
- molecules of diff sizes which can enter bead are eluted w/ Ve
What can affect elution vol (Ve), and what does this mean?
- flow rate
- buffer composition
- so calibration procedure and experiment should be carried out under same conditions
What is whole vol of liquid in column (Vl)?
- elution vol of smallest molecules which can completely penetrate beads?
How is vol of liquid inside beads (Vi) calc?
- Vl - Vo
How is total column vol (Vt) calc?
- Vo + Vi + Vs
What is vol of solid beads (Vs) and what is its normal value?
- varies for diff types of gel filtration matrices and falls in range between 5% and 15% of total column vol?
What are some of the practical uses of gel filtration?
- desalting or buffer exchange
- purification of macromolecules
- analysis of oligomeric state of protein and protein complexes (what we did)
What gel filtration matrix did we use, and what separation range did this provide?
- Sephadex G100 superfine
- separating range = 4000Da to 100,000 Da
What improvement was dev to Sephradex matrix, and what does it involve?
- cross linked agarose beads w/ large pores filled w/ dextran dictating final pore size
- adv is high res w/ short run times and good recovery
How are calibration plots made for gel filtration experiments?
- plot elution vol versus size
- in practise use molecular weight as measure of size of molecules
- but linear relationship between elution vol (Ve) and logMW
- most commonly do Kav vs logMW
What is a Kav value, and how are they calc?
- proportion of vol inside beads available for a given vol
- (Ve - Vo) / (Vt - Vo)
Does Sephradex have good separation power?
- no, relatively poor
What is important during a gel filtration experiment?
- columns must be kept vertical
- avoid disturbances at top of column during sample app
- don't touch frit when applying sample
- do not change positions of any components and don't let column run dry
What can gel filtration be used for?
- purify proteins
- determine molecular mass and subunit composition of oligomeric proteins
What is SDS-PAGE used for?
- analyse extent of purity of protein sample and to estimate molecular size of proteins in solution
What is affinity chromatography used for?
- powerful way to select for correctly folded proteins
What can be used to monitor efficiency at each step of purification process?
- calc of enzyme activity and its value per mg of protein (specific activity)
What is enzyme activity, and what are its usual units?
- 1 unit of activity is amount of enzyme needed to convert 1 μmole of substrate to product in 1 min
How is specific activity calc, and what are its usual units?
- enz activity / protein conc
How is total activity calc, and what does it provide?
- enzyme activity x vol of sample
- provides total no. units in sample at this stage
How is % yield calc?
- (total activity after / total activity before) x100
How is a chromatogram generally plotted?
- vol on X-axis (marking elution fractions against approp vol)
- absorbance on Y-axis
- work from right to left when plotting peaks
- harder when lack of points and protein can aggregate non-specifically so some peaks close together
What properties other than MW can affect way protein travels down gel filtration column?
- shape --> globular proteins travel diff to fibrous, which can get into smaller pores than MW would suggest
- affinity for column matrix itself
- fast eq between diff oligomeric states of protein, eg. dimer and tetramer forms
What effects do other properties affecting travel down column have on use of column?
- results need to be treated w/ caution and verified by another technique
- eg. analytical ultracentrifugation or dynamic light scattering
What are the basic principles of SDS-PAGE?
- denature protein w/ heat and anionic detergent SDS
- coat protein in -ve charge from SDS to give uniform charge density
- load sample onto polyacrylamide gel and apply electric field
- protein samples travel towards +ve electrode
- "sieve" proteins through acrylamide matrix and separate out on basis of size
- small proteins travel faster and further into gel
- stain gel and compare proteins under analysis w/ set of known MW markers
Why might result in estimating MW of Hb be diff from gel filtration and SDS-PAGE experiments?
- SDS-PAGE denatures protein sample and breaks up any higher order quaternary structures, so shows just monomer MW
- gel filtration should leave it intact, so show full MW
What can comparing SDS-PAGE and gel filtration results tell you?
- indication of stoichiometry of protein in solution, ie. how many copies of each polypeptide chain are present
What are some chromatographic methods involving direct interaction of proteins w/ column?
- ion exchange chromatography
- hydrophobic interaction chromatography
- affinity chromatography
What does ion exchange chromatography (IEC) separate molecules according to, and how?
- net surface charge
- typically through ionic interactions between charged amino acid side chains and surface charge of ion exchange matrix
What is the isoelectric point (pI), and what do the diff values mean?
- pH at which net charge of protein is 0
- >7 are basic proteins
- <7 are acidic proteins
- if pI = 7 then neutral
How does ion exchange chromatography work?
- pI used to describe net charge of protein
- under right pH and low salt conditions, a protein can be bound to a column and then eluted by increasing salt concs or by changing pH
What are the 2 types of ion exchange chromatography?
- anion exchange chromatography --> uses +vely charged functional groups to capture -vely charged proteins
- cation exchange chromatography --> uses -vely charged functional groups to capture +vely charged proteins
How does hydrophobic interaction chromatography (HIC)?
- hydrophobic residues exposed on surface of protein to some extent, so can interact w/ hydrophilic groups (phenyl/butyl/ether) under certain conditions
- for protein to bind to HIC matrix water must be removed from surface using high concs of certain salts (often ammonium sulphate)
- proteins then eluted when salt conc decreased
Why is affinity chromatography the most powerful method of chromatography?
- based on specific binding
What is the problem with affinity chromatography and how was this overcome?
- for each enzyme specific matrix has to be created, often difficult, time consuming or impossible
- dev of tagged proteins
How are tags used in affinity chromatography, and what are the most common tags?
- genetically attached to a protein by cloning gene of interest into specialised plasmid, and resulting fusion protein expressed in bacterial cells
- His6, GST, MBP
What are the principles of pseudo-affinity chromatography?
- ligand attached to matrix similar in structure to substrate
- commonly used matrix is cross-linked Heparin (eg. Heparin-Sepharose), used to purify DNA-binding proteins as heparin similar to phosphate chain in DNA
What are the principles of dye-chromatography?
- variant of pseudo-affinity chromatography
- ligands formed by synthetic polycyclic dyes
- ligands show certain structural similarities to cofactors NAD(H)/NADP(H), so widely used for purification of deHase, kinases etc.
What is the procedure for GDH purification using a column packed w/ Remazol-Sepharose?
- app of CFE onto column to allow GDH to bind to matrix
- washing out any unbound material
- elution of bound GDH
--> by NAD(H) = biospecific, gives high purity, but high conc req often too expensive
--> or high salt conc = nonspecific, but matrix highly selective so purity still quite high
How is purification factor calc?
- specific activity after / specific activity before
What does it mean to equilibrate a column?
- set the conditions
When trying to purify GDH why was there an initial peak in the chromatogram and why was there only 1 other peak?
- 1st is majority of proteins in CFE
- GDH specifically selected and rest washed off
What type of enzyme does Remazol select for?
- active (folded) enzymes
If further purification is req of an IEC_HIC_SEC sample what other method could be done?
- 2nd IEC column of opp type
- chromatofocussing column
- antibody column
- salt precipitation step
If further purification was req was req of GDH prep from Remazol column, what kind of chromatography would be best?
- size exclusion
- highest yield and purification factor
What genetic and biochemical approach could be used to look at whether proteins interact w/ each other?
- genetic = two-hybrid
- biochemical = pull down assay
Which protein interacts w/ Y14?
What are the general conditions for design of primers?
- choose restriction sites which are not present in insert, as do not want to cut this
- keep restriction sites in frame, others will change downstream seq
- only choose 1 restriction site w/in plasmid, and if have choice of more than 1, then choose 1 that minimises extra AAs
- if tag at 3' end don't leave stop codon as tag won't be translate, but if tag at 5' end leave it
- directed cloning is using diff enzymes in each oligo
- if problem w/ enzymes choice in plasmid and insert, then restrict each w/ diff enzymes but gen compatible ends
What are the principles of the 2-hybrid system?
- if prey not complementary to bait don't bind, RNA pol not brought to promoter, so lacZ gene not expressed and colonies stay white
- if prey and bait can bind, then RNA pol attracted to promoter, so lacZ gene expressed and colonies appear blue
What is the role of carrying out transformation efficiencies, and what result would you expect from them?
- to check cells are competent
How is transformation efficiency calc?
- no. CFU / μg DNA
What resistance genes did bait plasmid, prey plasmids and reporter gene cassette have?
- bait plasmids (pBD) = chloramphenicol
- prey plasmids (pAR) = ampicillin
- reporter gene cassette = kanamycin resistance gene
Why was the reporter strain used chosen?
- highly competent for transformation
- harbours reporter gene cassette
What -ve controls were used in co-transformations?
- pBD + pAR (make sure prey plasmid didn't interact w/ bait plasmid w/o protein present)
- pBD-Y14 + pAR (make sure prey plasmid didn't interact w/ Y14)
- pBD + pAR-X (make sure it's Y14 and not bait plasmid that interacts w/ protein X)
- pBD + pAR-Y (make sure it's Y14 and not bait plasmid that interacts w/ protein Y)
What 2 co-transformations were carried out to find which protein interacts w/ Y14?
- pBD-Y14 + pAR-X
- pBD-Y14 + pAR-Y
What are the advantages of bacterial 2-hybrid (in prok host cell) compared to yeast 2-hybrid (in euk host cell)?
- bacteria grow much faster than yeast (4/5x)
- bacteria easier to transform
- yeast could interact w/ other proteins, whereas bacteria wouldn't as never seen it before
What are the disadvantages of bacterial 2-hybrid (in prok host cell) compared to yeast 2-hybrid (in euk host cell)?
- bacteria can have low expression levels due to codon usage (species tend to favour 1 codon when there are multiple for the same AAs), so don't have enough of correct tRNA available
- bacteria lack ability to give post-translational mods, may be req for euk proteins to function
What are the advantages of using affinity purification, over other methods of protein purification?
- engineering selective property into your protein by means of a tag, rather than relying on intrinsic properties to separate them
- pot allows large amounts of protein to be prod, then easily and cleanly separated from other proteins
How is affinity purification carried out?
- tagged protein prod
- for this bacteria harbouring plasmid expressing tagged protein grown to log phase to give reasonable cell density
- expression of tagged gene induced and culture incubated to allow accum of tagged protein
- bacterial cells lysed, releasing cell contents (WCL)
- insoluble cell debris removed by centrifugation
- resulting solution contains all soluble cellular proteins, inc tagged protein = CFE
- tagged protein then affinity purified from CFE
In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after centrifugation of WCL to obtain CFE?
- GST-Y14 = supernatant
- soluble = supernatant
- insoluble = in pellet after centrifugation
In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after binding of CFE to beads?
- GST-Y14 = bound to beads
- soluble = supernatant
- insoluble = not present
In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after 1st and 2nd wash of above beads w/ buffer?
- GST-Y14 = bound to beads
- soluble = some in supernatant but less present after each wash
- insoluble = not present
In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after 3rd wash of beads w/ buffer?
- GST-Y14 = bound to beads
- soluble = not present
- insoluble = not present
In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after incubating beads w/ buffer containing glutathione?
- GST-Y14 = supernatant
- soluble = not present
- insoluble = not present
What are the characteristics of a polyacrylamide gel?
- creates finer mesh than agarose
- req free radicals to attach acrylamide monomers
- gel composed of polyacrylamide chains which forms lattice
What is the purpose of a miniprep?
- extract and prepare plasmid DNA
What is the role of SDS?
- detergent that causes bacterial cell wall to lyse by solubilising protein and lipids in cell wall
- allowing isolation of plasmid DNA
Why do we have to re-centrifuge the empty column after we have washed it w/ DNA wash buffer?
- remove residual ethanol
- other impurities removed by wash stages
- ethanol could inhibit downstream apps using plasmid DNA, such as restriction digests and transformations
Why can it be difficult to accurately predict the size of plasmid from agarose gel?
- supercoiled plasmids migrate faster than linear/circular as encounters less resistance from gel matrix
What might influence the yield of plasmid from minipreps?
- plasmids copy no. and size
- how cell culture has grown
- cell culture vol
- host strain used
- efficiency of kit to purify plasmid DNA
Why might co-transformations result in fase +ves?
- overexpression of bait and prey proteins could force interactions that would not naturally occur in cell
- unnatural mods and folding of proteins in bacteria could cause binding
- bait protein may have transcriptional activity on its own, leading to expression of reporter gene
- prey protein may have DNA binding activity on its own, leading to expression of reporter gene
What are the necessary features of a pair of bacterial 2-hybrid plasmids?
- 2 plasmids must either express bait protein fused to DNA binding domain or prey protein fused to activating region of RNA pol --> need both DNA binding domain and RNA pol subunit to get expression of reporter gene
- plasmids confer diff antibiotic resistances so can select for presence of both plasmids in E. coli
- diff origins of rep prevent competition for the hosts rep machinery which could result in only 1 plasmid being maintained in E. coli, best to only co-transform plasmids from diff origin ("incompatibility groups")
What are the 3 roles of SDS page loading buffer?
- denatures proteins (SDS and other reducing agents)
- glycerol to make sink at bottom of well
- dye (bromophenol blue) to visualise movement through gel
What are the principles of pull down assays?
- reveal presence of direct protein:protein interactions
- exploit affinity purification, in that bait protein is affinity tagged, so its interactions w/ other prey proteins can be observed via their co-purification
- during wash steps, prey protein remains bound to bait protein, and non-interacting proteins removed
- prey protein present w/ bait after elution
- bait protein "pulls down" prey protein from reaction
What does choosing conditions for crystallography involve?
- exact combo of reagents, pH, temp, counter ions, substrates, inhibitors etc.
What are common reagents for crystallisation?
- polyethylene glycols of various MWs
- ammonium sulphate
What values of pH are usually tested for X-ray crystallisation?
- occasionally more extreme values successful, even though outside normal range found in biological systems
Why can metal ions be a vital addition to crystallisation conditions?
- large no. proteins bind metal ions for their function in catalysis or structural capacity
How can the presence/absence of a cofactor being bound affect crystallisation?
- can cause changes to conformation
- resultant alt shape may be essential for crystallisation, as could allow formation of contacts between molecules in crystal lattice
Why is a preliminary screening process always needed in crystallisation?
- narrow down search from many 1000s poss combos
What is the most common method of crystallography?
- vapour diffusion
How does vapour diffusion work?
- generally, high vapour pressure in cup, so water evaporates from cup
- conc of the reagents increases until equilibrated
- protein conc also increasing
- adjust conditions slowly so protein crosses solubility limit and emerges in crystalline state
What happens to a protein when it reaches point at which it becomes insoluble?
- either precipitates out as amorphous largely aggregated mass or adopt ordered crystalline state
How are crystals effectively an amplification system?
- 1000s copies aligned w/ each other in same orientation, forming 3D structure
What is the problem w/ having protein sample in 20% w/v NaCl, and how could this be solved?
- too high (we used 1-11%)
- more NaCl disrupts H bonds, so crystal wont form
- could lower through dialysis (only salt moves through holes) or dilute
Why is it difficult to determine the structure of an ES complex?
- converted to enz and product quickly
- reaction occurring and rearrangment of bonds mean can't crystallise it before reaction is over
- so get mix of enz, sub and product
- non-homogenous mixture harder to crystallise and interpret
How could the problem of obtaining ES complexes be overcome, and what is the problem w/ these methods?
- low temp to slow reaction
- substrate analog
- non competitive inhibitor
- use mutated, catalytically inactive enz
- change pH to be less optimal
- leave out essential cofactor, eg. metal ion
- carry out quick soaking experiment on preformed crystals
- problem = all risk enz not being in fully correct formation that occurs in vivo
Why are lysozyme crystals so fragile?
- around 50% solvent, so atoms only only just touching
What is the advantage of the presence of solvent in lysozyme crystals?
- offers opportunity to soak things into crystals which couldn't be added beforehand (as would affect crystal formation)
If carried out a set of experiments that after 1 week had only clear drops, what
might be some poss problems, and what would you do next to try to obtain crystals?
- precipitant conc too low at pHs tested
- protein conc not high enough
- protein not pure enough, or been degraded (proteolysis or unfolding)
- change precipitant, its conc, pH, improve purity, add protease inhibitors, change temp
What happens to a crystal when transferred to air, water or dye solution, and why?
- air = dried out, as needs to be kept in liquid to stop water evaporation from large solvent channels, which breaks contacts w/ subsequent collapse of crystal channels
- water = dissolved, as decreases effective conc of protein to below solubility limit and water competes for contacts
- dye = turned deeper blue than surroundings, as channels mean any small molecule can diffuse down them and bind to sites on protein, and not just coat outside
Why are structures from standard transfer and soaking procedure sometimes diff for drugs bound to proteins, than when co-crystallised together?
- protein cannot change it shape fully or at all due to lattice
- drug cannot access target site in same way as other features are in the way when proteins adopted conformation for crystallisation
- other variables in crystallisation, eg. certain ions or pH, might alt binding site
Why is an automated system req for efficient crystallisation screening?
- v time consuming by hand
- for new protein where conditions unknown, may need over 500 initial trials
- would req lots of protein
- automated can use v small vols repetitively and quickly
How does efficient crystallisation make use of automated liquid handing, and what must these robots be capable of?
- dispense v small vols (0.05 - 0.2 µl) of protein and matching amounts of crystallising reagent v accurately and precisely into small cups
- ideally should also dispense larger vols (25 - 100 µl) of crystallisation reagent into wells
- must accom issues of viscosity of samples, consistent repetitive dispensing, cleaning between protein samples/screens etc.
How are growing crystals kept under carefully controlled temps after trays set up, and why is this important?
- exact temp variable and pot critical as affects protein solubility
- typically constant value varying by less than 1°, near room temp
- regular inspection and various devices based around coupled microscope and camera systems
How long does it take for crystallisation to occur, and what does this mean?
- so repeat obs req to see any changes in drops
How are X-ray diffraction studies carried out, after crystals grown?
- crystals harvested individually in fire loops and mounted on goniostats for precise placement in fine X-ray beam, prod by generator
- alignment of crystal w/ beam critical, as is ability to rotate crystal to capture all poss diffracted rays
- all defracted X-ray beams collected on detector systems
- detection on phosphorent plate screened by laser, or on charged couples device (CCD) chip that allows direct readout
What does X-ray diffraction data give you?
- intensities and relevant positions of diffracted beams on detector and calc phases of X-ray waves
What do maps form X-ray diffraction show?
- distribution of e- density clouds around atoms
What is the final step in crystallisation experiments?
- examinations of maps and analysis of mol models fitted to them
What is hanging drop crystallisation?
- drop mixtures of protein and buffered crystallisation reagent suspended on underside of glass coverslips
- which are placed over reservoirs of buffered reagent
- also relies on vapour diffusion to equilibrate the conditions
What is the advantage of the hanging drop method?
- easy access to crystals for manipulation
What is the disadvantage of the hanging drop method?
- more complex to set up and not readily automated
What is the advantage of the sitting drop method?
- easy to prepare and readily automated
What is the disadvantage of the sitting drop method?
- access to crystals more difficult
Why might ethylene glycol be added to crystallisation experiment?
- antifreeze to "cryoprotect" crystals and stop water ice crystals forming, which damage protein crystal and disrupt packing between molecules in lattice
What are the small spherical patches of e- density seen on X-ray diffraction maps?
- water molecules --> only see oxygens
What are most new drugs dev from?
- antibody based
- or variants of natural products (w/ improved potency, solubility etc.)