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MBB265 > Semester 2 > Flashcards

Semester 2 Flashcards

1
Q

How does gel filtration work?

A
  • 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
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2
Q

What is the vol of eluent proportional to in gel filtration?

A
  • time taken for collection
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3
Q

What is the result of using small beads in gel filtration matrix?

A
  • less diffusion, so elution peaks sharper and separation better?
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4
Q

What is void volume (Vo)?

A
  • molecules excluded from beads elution vol, equal to liquid outside beads
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5
Q

What is elution volume (Ve)?

A
  • molecules of diff sizes which can enter bead are eluted w/ Ve
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6
Q

What can affect elution vol (Ve), and what does this mean?

A
  • flow rate
  • buffer composition
  • temp
  • so calibration procedure and experiment should be carried out under same conditions
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7
Q

What is whole vol of liquid in column (Vl)?

A
  • elution vol of smallest molecules which can completely penetrate beads?
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8
Q

How is vol of liquid inside beads (Vi) calc?

A
  • Vl - Vo
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9
Q

How is total column vol (Vt) calc?

A
  • Vo + Vi + Vs
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10
Q

What is vol of solid beads (Vs) and what is its normal value?

A
  • varies for diff types of gel filtration matrices and falls in range between 5% and 15% of total column vol?
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11
Q

What are some of the practical uses of gel filtration?

A
  • desalting or buffer exchange
  • purification of macromolecules
  • analysis of oligomeric state of protein and protein complexes (what we did)
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12
Q

What gel filtration matrix did we use, and what separation range did this provide?

A
  • Sephadex G100 superfine

- separating range = 4000Da to 100,000 Da

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13
Q

What improvement was dev to Sephradex matrix, and what does it involve?

A
  • Superdex
  • 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
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14
Q

How are calibration plots made for gel filtration experiments?

A
  • 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
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15
Q

What is a Kav value, and how are they calc?

A
  • proportion of vol inside beads available for a given vol

- (Ve - Vo) / (Vt - Vo)

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16
Q

Does Sephradex have good separation power?

A
  • no, relatively poor
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17
Q

What is important during a gel filtration experiment?

A
  • 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
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18
Q

What can gel filtration be used for?

A
  • purify proteins

- determine molecular mass and subunit composition of oligomeric proteins

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19
Q

What is SDS-PAGE used for?

A
  • analyse extent of purity of protein sample and to estimate molecular size of proteins in solution
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20
Q

What is affinity chromatography used for?

A
  • powerful way to select for correctly folded proteins
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21
Q

What can be used to monitor efficiency at each step of purification process?

A
  • calc of enzyme activity and its value per mg of protein (specific activity)
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22
Q

What is enzyme activity, and what are its usual units?

A
  • 1 unit of activity is amount of enzyme needed to convert 1 μmole of substrate to product in 1 min
  • units/ml
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23
Q

How is specific activity calc, and what are its usual units?

A
  • enz activity / protein conc

- units/mg

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24
Q

How is total activity calc, and what does it provide?

A
  • enzyme activity x vol of sample

- provides total no. units in sample at this stage

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25
Q

How is % yield calc?

A
  • (total activity after / total activity before) x100
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26
Q

How is a chromatogram generally plotted?

A
  • 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
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27
Q

What properties other than MW can affect way protein travels down gel filtration column?

A
  • 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
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28
Q

What effects do other properties affecting travel down column have on use of column?

A
  • results need to be treated w/ caution and verified by another technique
  • eg. analytical ultracentrifugation or dynamic light scattering
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29
Q

What are the basic principles of SDS-PAGE?

A
  • 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
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30
Q

Why might result in estimating MW of Hb be diff from gel filtration and SDS-PAGE experiments?

A
  • 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
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31
Q

What can comparing SDS-PAGE and gel filtration results tell you?

A
  • indication of stoichiometry of protein in solution, ie. how many copies of each polypeptide chain are present
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32
Q

What are some chromatographic methods involving direct interaction of proteins w/ column?

A
  • ion exchange chromatography
  • hydrophobic interaction chromatography
  • affinity chromatography
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33
Q

What does ion exchange chromatography (IEC) separate molecules according to, and how?

A
  • net surface charge

- typically through ionic interactions between charged amino acid side chains and surface charge of ion exchange matrix

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34
Q

What is the isoelectric point (pI), and what do the diff values mean?

A
  • pH at which net charge of protein is 0
  • > 7 are basic proteins
  • <7 are acidic proteins
  • if pI = 7 then neutral
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35
Q

How does ion exchange chromatography work?

A
  • 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
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36
Q

What are the 2 types of ion exchange chromatography?

A
  • 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
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37
Q

How does hydrophobic interaction chromatography (HIC)?

A
  • 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
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38
Q

Why is affinity chromatography the most powerful method of chromatography?

A
  • based on specific binding
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39
Q

What is the problem with affinity chromatography and how was this overcome?

A
  • for each enzyme specific matrix has to be created, often difficult, time consuming or impossible
  • dev of tagged proteins
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40
Q

How are tags used in affinity chromatography, and what are the most common tags?

A
  • genetically attached to a protein by cloning gene of interest into specialised plasmid, and resulting fusion protein expressed in bacterial cells
  • His6, GST, MBP
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41
Q

What are the principles of pseudo-affinity chromatography?

A
  • 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
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42
Q

What are the principles of dye-chromatography?

A
  • 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.
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43
Q

What is the procedure for GDH purification using a column packed w/ Remazol-Sepharose?

A
  • 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
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44
Q

How is purification factor calc?

A
  • specific activity after / specific activity before
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45
Q

What does it mean to equilibrate a column?

A
  • set the conditions
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46
Q

When trying to purify GDH why was there an initial peak in the chromatogram and why was there only 1 other peak?

A
  • 1st is majority of proteins in CFE

- GDH specifically selected and rest washed off

47
Q

What type of enzyme does Remazol select for?

A
  • active (folded) enzymes
48
Q

If further purification is req of an IEC_HIC_SEC sample what other method could be done?

A
  • 2nd IEC column of opp type
  • chromatofocussing column
  • antibody column
  • salt precipitation step
49
Q

If further purification was req was req of GDH prep from Remazol column, what kind of chromatography would be best?

A
  • size exclusion

- highest yield and purification factor

50
Q

What genetic and biochemical approach could be used to look at whether proteins interact w/ each other?

A
  • genetic = two-hybrid

- biochemical = pull down assay

51
Q

Which protein interacts w/ Y14?

A
  • MAGOH
52
Q

What are the general conditions for design of primers?

A
  • 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
53
Q

What are the principles of the 2-hybrid system?

A
  • DIAG*
  • 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
54
Q

What is the role of carrying out transformation efficiencies, and what result would you expect from them?

A
  • to check cells are competent

- white

55
Q

How is transformation efficiency calc?

A
  • no. CFU / μg DNA
56
Q

What resistance genes did bait plasmid, prey plasmids and reporter gene cassette have?

A
  • bait plasmids (pBD) = chloramphenicol
  • prey plasmids (pAR) = ampicillin
  • reporter gene cassette = kanamycin resistance gene
57
Q

Why was the reporter strain used chosen?

A
  • highly competent for transformation

- harbours reporter gene cassette

58
Q

What -ve controls were used in co-transformations?

A
  • 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)
59
Q

What 2 co-transformations were carried out to find which protein interacts w/ Y14?

A
  • pBD-Y14 + pAR-X

- pBD-Y14 + pAR-Y

60
Q

What are the advantages of bacterial 2-hybrid (in prok host cell) compared to yeast 2-hybrid (in euk host cell)?

A
  • 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
61
Q

What are the disadvantages of bacterial 2-hybrid (in prok host cell) compared to yeast 2-hybrid (in euk host cell)?

A
  • 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
62
Q

What are the advantages of using affinity purification, over other methods of protein purification?

A
  • 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
63
Q

How is affinity purification carried out?

A
  • 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
64
Q

In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after centrifugation of WCL to obtain CFE?

A
  • GST-Y14 = supernatant
  • soluble = supernatant
  • insoluble = in pellet after centrifugation
65
Q

In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after binding of CFE to beads?

A
  • GST-Y14 = bound to beads
  • soluble = supernatant
  • insoluble = not present
66
Q

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?

A
  • GST-Y14 = bound to beads
  • soluble = some in supernatant but less present after each wash
  • insoluble = not present
67
Q

In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after 3rd wash of beads w/ buffer?

A
  • GST-Y14 = bound to beads
  • soluble = not present
  • insoluble = not present
68
Q

In affinity purification, where are GST-Y14, other soluble cellular proteins and insoluble cellular protein/cell debris, after incubating beads w/ buffer containing glutathione?

A
  • GST-Y14 = supernatant
  • soluble = not present
  • insoluble = not present
69
Q

What are the characteristics of a polyacrylamide gel?

A
  • creates finer mesh than agarose
  • req free radicals to attach acrylamide monomers
  • gel composed of polyacrylamide chains which forms lattice
70
Q

What is the purpose of a miniprep?

A
  • extract and prepare plasmid DNA
71
Q

What is the role of SDS?

A
  • detergent that causes bacterial cell wall to lyse by solubilising protein and lipids in cell wall
  • allowing isolation of plasmid DNA
72
Q

Why do we have to re-centrifuge the empty column after we have washed it w/ DNA wash buffer?

A
  • remove residual ethanol
  • other impurities removed by wash stages
  • ethanol could inhibit downstream apps using plasmid DNA, such as restriction digests and transformations
73
Q

Why can it be difficult to accurately predict the size of plasmid from agarose gel?

A
  • supercoiled plasmids migrate faster than linear/circular as encounters less resistance from gel matrix
74
Q

What might influence the yield of plasmid from minipreps?

A
  • plasmids copy no. and size
  • how cell culture has grown
  • cell culture vol
  • host strain used
  • efficiency of kit to purify plasmid DNA
75
Q

Why might co-transformations result in fase +ves?

A
  • 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
76
Q

What are the necessary features of a pair of bacterial 2-hybrid plasmids?

A
  • 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”)
77
Q

What are the 3 roles of SDS page loading buffer?

A
  • denatures proteins (SDS and other reducing agents)
  • glycerol to make sink at bottom of well
  • dye (bromophenol blue) to visualise movement through gel
78
Q

What are the principles of pull down assays?

A
  • 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
79
Q

What does choosing conditions for crystallography involve?

A
  • exact combo of reagents, pH, temp, counter ions, substrates, inhibitors etc.
80
Q

What are common reagents for crystallisation?

A
  • polyethylene glycols of various MWs
  • ammonium sulphate
  • alcohols
81
Q

What values of pH are usually tested for X-ray crystallisation?

A
  • 5-9

- occasionally more extreme values successful, even though outside normal range found in biological systems

82
Q

Why can metal ions be a vital addition to crystallisation conditions?

A
  • large no. proteins bind metal ions for their function in catalysis or structural capacity
83
Q

How can the presence/absence of a cofactor being bound affect crystallisation?

A
  • can cause changes to conformation
  • resultant alt shape may be essential for crystallisation, as could allow formation of contacts between molecules in crystal lattice
84
Q

Why is a preliminary screening process always needed in crystallisation?

A
  • narrow down search from many 1000s poss combos
85
Q

What is the most common method of crystallography?

A
  • vapour diffusion
86
Q

How does vapour diffusion work?

A
  • 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
87
Q

What happens to a protein when it reaches point at which it becomes insoluble?

A
  • either precipitates out as amorphous largely aggregated mass or adopt ordered crystalline state
88
Q

How are crystals effectively an amplification system?

A
  • 1000s copies aligned w/ each other in same orientation, forming 3D structure
89
Q

What is the problem w/ having protein sample in 20% w/v NaCl, and how could this be solved?

A
  • 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
90
Q

Why is it difficult to determine the structure of an ES complex?

A
  • 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
91
Q

How could the problem of obtaining ES complexes be overcome, and what is the problem w/ these methods?

A
  • 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
92
Q

Why are lysozyme crystals so fragile?

A
  • around 50% solvent, so atoms only only just touching
93
Q

What is the advantage of the presence of solvent in lysozyme crystals?

A
  • offers opportunity to soak things into crystals which couldn’t be added beforehand (as would affect crystal formation)
94
Q

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?

A
  • 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
95
Q

What happens to a crystal when transferred to air, water or dye solution, and why?

A
  • 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
96
Q

Why are structures from standard transfer and soaking procedure sometimes diff for drugs bound to proteins, than when co-crystallised together?

A
  • 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
97
Q

Why is an automated system req for efficient crystallisation screening?

A
  • 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
98
Q

How does efficient crystallisation make use of automated liquid handing, and what must these robots be capable of?

A
  • 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.
99
Q

How are growing crystals kept under carefully controlled temps after trays set up, and why is this important?

A
  • 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
100
Q

How long does it take for crystallisation to occur, and what does this mean?

A
  • varies

- so repeat obs req to see any changes in drops

101
Q

How are X-ray diffraction studies carried out, after crystals grown?

A
  • 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
102
Q

What does X-ray diffraction data give you?

A
  • intensities and relevant positions of diffracted beams on detector and calc phases of X-ray waves
103
Q

What do maps form X-ray diffraction show?

A
  • distribution of e- density clouds around atoms
104
Q

What is the final step in crystallisation experiments?

A
  • examinations of maps and analysis of mol models fitted to them
105
Q

What is hanging drop crystallisation?

A
  • 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
106
Q

What is the advantage of the hanging drop method?

A
  • easy access to crystals for manipulation
107
Q

What is the disadvantage of the hanging drop method?

A
  • more complex to set up and not readily automated
108
Q

What is the advantage of the sitting drop method?

A
  • easy to prepare and readily automated
109
Q

What is the disadvantage of the sitting drop method?

A
  • access to crystals more difficult
110
Q

Why might ethylene glycol be added to crystallisation experiment?

A
  • antifreeze to “cryoprotect” crystals and stop water ice crystals forming, which damage protein crystal and disrupt packing between molecules in lattice
111
Q

What are the small spherical patches of e- density seen on X-ray diffraction maps?

A
  • water molecules –> only see oxygens
112
Q

What are most new drugs dev from?

A
  • antibody based

- or variants of natural products (w/ improved potency, solubility etc.)

113
Q

Why are natural products the best option for dev of new drugs?

A
  • MOs and plants have evolved 2° metabolic pathways for synthesis of compounds that higher organisms can’t synthesise

MBB265 (3 decks)

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