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Chapter 2 Strategies for Protein Separation •A mammalian cell has 50,000-100,000 proteins (including PTMs) •Selective techniques isolate one (affinity) • Non-selective techniques isolate large number/all •Proteomics require isolation of proteins at high resolution (selective) •Must be amenable to high throughput (HT) technology 1 Proteins can be isolated on the grounds of the following properties: •Mass/density (hydrodynamic) •Size (gel filtration) •pI (pI precipitation of IEF) •Charge (Ion-exchange) •Solubility (acetone of ammoniumsulfate ppt) •Structure (affinity) Proteins differ by size myoglobin myosin insulin (β β-chain) 2 Proteins have different shapes Human insulin (β β-chain) Gal4 homodimer Proteins have charges Ribbon Electrostatic surface 3 Molecular movement in an electric field - + + ν= Eq f •ν Is velocity of the molecule •E is electeric field strength (volts/cm •q is net charge on molecule •f is frictional coefficient Aliphatic amino acids (VAGLIP) Glycine, Gly, G no charge Hydrophobicity = 0.67 MW 57Da pKa COOH = 2.35 pKa NH2 = 9.6 pI=5.97 Alanine, Ala, A no charge Hydrophobicity = 1.0 MW 71Da pKa COOH = 2.34 pKa NH2 = 9.69 pI = 6.01 Valine, Val, V no charge Hydrophobicity = 2.3 MW 99Da pKa COOH = 2.32 pKa NH2 = 9.62 pI = 5.97 Leucine, Leu, L no charge Hydrophobicity = 2.2 MW 113Da pKa COOH = 2.36 pKa NH2 = 9.60 pI = 5.98 Isoleucine, Ile, I no charge Hydrophobicity = 3.1 MW 113Da pKa COOH = 2.36 pKa NH2 = 9.68 pI = 6.02 Proline, Pro, P no charge Hydrophobicity = -0.29 MW 97Da pKa COOH = 1.99 pKa NH2 = 10.96 pI = 6.48 4 Aromatic amino acids (FYW) Phenylalanine, Phe, F no charge Hydrophobicity = 2.5 Absorbs UV MW 147Da pKa COOH = 1.83 pKa NH2 = 9.13 pI=5.48 Tyrosine, Tyr, Y weak charge Hydrophobicity = 0.08 Absorbs UV MW 163Da pKa COOH = 2.20 pKa NH2 = 9.11 pI=5.66 Tryptophan, Trp, W no charge Hydrophobicity = 1.5 Absorbs UV MW 186Da pKa COOH = 2.38 pKa NH2 = 9.39 pI=5.89 Polar but uncharged (SNQT) Serine, Ser, S no charge Hydrophobicity = -1.1 MW 87Da pKa COOH = 2.21 pKa NH2 = 9.15 pI = 5.68 Threonine, Thr, T no charge Hydrophobicity = -0.75 MW 101Da pKa COOH = 2.11 pKa NH2 = 9.62 pI = 5.87 Asparagine, Asn, N no charge Hydrophobicity = -2.7 MW 114Da pKa COOH = 2.02 pKa NH2 = 8.08 pI = 5.41 Glutamine, Gln, Q no charge Hydrophobicity = -2.9 MW 128Da pKa COOH = 2.17 pKa NH2 = 9.13 pI = 5.65 5 Sulphur containing (CM) Cysteine, Cys, C weak charge Hydrophobicity = 0.17 MW 103Da pKa COOH = 1.96 pKa NH2 = 8.18 pI = 5.07 Methionine, Met, M no charge Hydrophobicity = 1.1 MW 131Da pKa COOH = 2.28 pKa NH2 = 9.21 pI = 5.74 Charged (DEHKR) Acidic Aspartic acid, Asp, D negative charge Hydrophobicity = -3.0 MW 115Da pKa COOH = 2.19 pKa NH2 = 9.60 pI = 2.77 Glutamic acid, Glu, E negative charge Hydrophobicity = -2.6 MW 129Da pKa COOH = 2.19 pKa NH2 = 9.67 pI = 3.22 Basic Histidine, His, H Weak positive charge Hydrophobicity = -1.7 MW 137Da pKa COOH = 1.82 pKa NH2 = 9.17 pI = 7.59 Lysine, Lys, K positive charge Hydrophobicity = -4.6 MW 128Da pKa COOH = 2.18 pKa NH2 = 8.95 pI = 9.47 Arginine, Arg, R positive charge Hydrophobicity = -7.5 MW 156Da pKa COOH = 2.17 pKa NH2 = 9.04 pI = 10.76 6 Amino acid pKa values A.A. Carboxylic acid Amine Side Chain A 2.3 9.9 - C 1.8 10.8 8.6 D 2.0 10.0 4.5 E 2.2 9.7 4.5 F 1.8 9.1 - G 2.4 9.8 6.8 H 1.8 9.2 I 2.4 9.7 - K 2.2 9.2 11.1 L 2.4 9.6 - M 2.3 9.2 - N 2.0 8.8 - P 2.0 10.6 - Q 2.2 9.1 - R 1.8 9.0 12.5 S 2.1 9.2 - T 2.6 10.4 - V 2.3 9.6 - W 2.4 9.4 - Y 2.2 9.1 9.8 The Henderson-Hasselbalch equation HA H+ + A- (1) Ka = [H+][A-]/[HA] (2) [H+] = Ka[HA]/[A-] (3) - log[H+] = - logKa - log[HA]/[A-] (4) pH = pKa + log[A-]/[HA] (5) pH = pKa + log(R) (6) pH - pKa = log(R) (7) 10(pH - pKa) = R (8) 7 Acid and base fractions in a titration [A-]/[HA] = R (1) [A-] = [HA]R (2) AT = [A-] + [HA] (3) Substitute eq. 2 in eq. 3 AT = [HA]R + [HA] = [HA](1 + R) [HA] = AT 1 (1 + R) Substitute rearranged eq. 2 in eq. 6 [A-] = AT R (1 + R) (4) (5) (6) (7) How to calculate the charge of an amino acid at a given pH value •Lets choose K •and lets choose pH 7.2 •K has three dissociable groups that can contribute to the charge: •an α-NH2, an α-COOH and an ε-NH2 group •The pKa values of the three groups are 9.2, 2.2 and 11.1 10(pH-pKa) = R Lets start with the amine: ∴10(7.2-9.2) = 10-2 Since the α–NH2 will carry a charge in the protonated –NH3+ form, we are interested in the HA species [HA] = AT(1/(1+R)) Assume AT = 1 ∴[HA] = 1/(1+ 10-2) = 1/1.01 = 0.99 Thus the α–NH2 is almost fully protonated at pH 7.2, and will contribute a charge of +0.99 to the amino acid 8 How to calculate the charge of an amino acid at a given pH value continued… •Lets look at the α–COOH next •The pKa value of the –COOH is 2.2 10(pH-pKa) = R ∴10(7.2-2.2) = 105 The dissociated –COO- is the charged species We are therefore interested in the A- species and not the HA [A-] = AT(R/(1+R)) Assume AT = 1 ∴ -] = 105/(1+ 105) = 100,000/100,001 = 0.999 ∴[A Thus the α-COOH is almost fully deprotonated at pH 7.2, and will contribute a charge of -0.999 to the amino acid How to calculate the charge of an amino acid at a given pH value continued… •Lets look at the ε–NH2 next •The pKa value of the ε–NH2 is 11.1 10(pH-pKa) = R ∴10(7.2-11.1) = 10-3.9 The protonated ε–NH3+ is the charged species We are therefore interested in the HA species and not the A[HA-] = AT(1/(1+R)) Assume AT = 1 ∴[HA] = 1/(1+ 10-3.9) = 1/1.0001259 = 0.999 Thus the ε–NH2 is almost fully protonated at pH 7.2, and will contribute a charge of +0.999 to the amino acid The net charge of lysine at pH7.2 is therefore the sum of the three charges: α-NH2 + α-COOH + ε–NH2 + 0.99 - 0.999 + 0.999 = + 0.99 9 Charge versus solution pH of cysteine 1. Calculate R for each dissociable group 2. Calculate [A-] or [HA] 3. Calculate fractional charge 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 COOH R 1.7 0.019952623 0.199526231 1.995262315 19.95262315 199.5262315 1995.262315 19952.62315 199526.2315 1995262.315 19952623.15 199526231.5 1995262315 19952623150 1.99526E+11 1.99526E+12 SH [A-] charge 0.019562304 0.166337531 0.666139425 0.952273279 0.995013121 0.999499064 0.999949884 0.999994988 0.999999499 0.99999995 0.999999995 0.999999999 1 1 1 -0.019562304 -0.166337531 -0.666139425 -0.952273279 -0.995013121 -0.999499064 -0.999949884 -0.999994988 -0.999999499 -0.99999995 -0.999999995 -0.999999999 -1 -1 -1 R 8.3 5.01187E-09 5.01187E-08 5.01187E-07 5.01187E-06 5.01187E-05 0.000501187 0.005011872 0.050118723 0.501187234 5.011872336 50.11872336 501.1872336 5011.872336 50118.72336 501187.2336 NH2 [A-] charge 5.01187E-09 5.01187E-08 5.01187E-07 5.01185E-06 5.01162E-05 0.000500936 0.004986879 0.047726721 0.333860575 0.833662469 0.980437696 0.998008711 0.999800514 0.999980048 0.999998005 -5.01187E-09 -5.01187E-08 -5.01187E-07 -5.01185E-06 -5.01162E-05 -0.000500936 -0.004986879 -0.047726721 -0.333860575 -0.833662469 -0.980437696 -0.998008711 -0.999800514 -0.999980048 -0.999998005 R 10.8 1.58489E-11 1.58489E-10 1.58489E-09 1.58489E-08 1.58489E-07 1.58489E-06 1.58489E-05 0.000158489 0.001584893 0.015848932 0.158489319 1.584893192 15.84893192 158.4893192 1584.893192 ! [HA] 1 1 0.999999998 0.999999984 0.999999842 0.999998415 0.999984151 0.999841536 0.998417615 0.984398338 0.863193111 0.38686318 0.059350943 0.006270012 0.000630559 charge 1 1 0.999999998 0.999999984 0.999999842 0.999998415 0.999984151 0.999841536 0.998417615 0.984398338 0.863193111 0.38686318 0.059350943 0.006270012 0.000630559 Total charge 0.980437691 0.833662419 0.333860073 0.047721693 0.004936604 -1.58489E-06 -0.004952611 -0.047880173 -0.33544246 -0.849264081 -1.11724458 -1.61114553 -1.94044957 -1.993710035 -1.999367445 " Titration curve of cysteine 1.0 0.5 0.0 Charge pH -0.5 -1.0 -1.5 -2.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH 10 Calculate the pH versus charge for a peptide The peptide DKG pKa = 9.8 H3N+ H O α C C H O H O α α N C C N C C O- pKa = 2.4 CH2 H CH2 COO- CH2 pKa = 3.9 CH2 H CH3 CH2 NH3+ pKa = 10.5 Charge versus solution pH of peptide DKG pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 N terminus pKa = 9.8 F charge R 1 1 0.999999998 0.999999998 0.999999984 0.999999984 0.999999842 0.999999842 0.999998415 0.999998415 0.999984151 0.999984151 0.999841536 0.999841536 0.998417615 0.998417615 0.984398338 0.984398338 0.863193111 0.863193111 0.38686318 0.38686318 0.059350943 0.059350943 0.006270012 0.006270012 0.000630559 0.000630559 6.30918E-05 6.30918E-05 Aspartic acid R-group Pka=3.9 F charge R 0.000125877 -0.000125877 0.001257343 -0.001257343 0.012432735 -0.012432735 0.11181577 -0.11181577 0.557311634 -0.557311634 0.926412444 -0.926412444 0.992119316 -0.992119316 0.999206302 -0.999206302 0.999920573 -0.999920573 0.999992057 -0.999992057 0.999999206 -0.999999206 0.999999921 -0.999999921 0.999999992 -0.999999992 0.999999999 -0.999999999 1 -1 Lysine R-group pKa=10.5 F charge R 1 1 1 1 0.999999997 0.999999997 0.999999968 0.999999968 0.999999684 0.999999684 0.999996838 0.999996838 0.999968378 0.999968378 0.999683872 0.999683872 0.996847691 0.996847691 0.96934657 0.96934657 0.759746927 0.759746927 0.240253073 0.240253073 0.03065343 0.03065343 0.003152309 0.003152309 0.000316128 0.000316128 C terminus pKa=2.9 F charge R 0.001257343 -0.001257343 0.012432735 -0.012432735 0.11181577 -0.11181577 0.557311634 -0.557311634 0.926412444 -0.926412444 0.992119316 -0.992119316 0.999206302 -0.999206302 0.999920573 -0.999920573 0.999992057 -0.999992057 0.999999206 -0.999999206 0.999999921 -0.999999921 0.999999992 -0.999999992 0.999999999 -0.999999999 1 -1 1 -1 Total charge 0.998616781 0.986309922 0.875751492 0.330872565 -0.483724394 -0.918534922 -0.99135724 -0.999443004 -1.003064939 -1.030644692 -1.2402522 -1.759746839 -1.969346561 -1.99684769 -1.999683872 You can also use the program “What charge?” to calculate charge versus pH curves of any amino acid, peptide or protein, based on the entered sequence. You can download the program from http://www.uovs.ac.za/faculties/documents/04/112/Software/11476-what_charge.zip 11 Titration curve of peptide DKG 1.0 0.5 Charge 0.0 -0.5 -1.0 -1.5 -2.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH SDS Page electrophoresis - - - - - - - SDS protein - - - - - - - - •Sodium dodecylsulfate (SDS) •Amphipatic molecule •0.1% SDS gives one SDS molecule per 2 amino acid residues •Thus charge per unit length of all proteins are the same •Proteins of the same size will have the same charge •Proteins separated mainly by size •Very basic proteins such as histones behave anomalously 12 Vertical slab gel apparatus Polyacrylamide percentage •The composition of polyacrylamide gels is expressed as: •%T grams acrylamide plus gram bis-acrylamide per 100ml solution •%C grams bisacrylamide per 100ml solution •Typical gels %T range from 3% to 20% •%C from 3 to 6% •The higher the acrylamide percentage, the smaller the gel pores •The higher the bis-acrylamide percentage, the samller the gel pores •Larger molecules cannot migrate through small gel pores •Percentage acrylamide has range within which molecules can be separated •3% gels for protein of 1000kDa •20% gels for proteins of 10kDa •Polyacrylamide gel electrophoresis = PAGE 13 Discontinuous SDS-PAGE glycine protein Cl- Stacking gel 3%T pH 6.8 Running gel 8-16%T pH8.8 •Sample buffer is •glycine •Cl•and protein •When glycine enters the gel, at pH6.8 it is a zwitterion •To maintain current, the Cland proteins migrate •The pores are very large, so the proteins can move fast •The glycine follows •This tends to concentrate the proteins in thin bands before they strike the running gel, where separation occurs •The glycine becomes charged in the running gel, and carries current with Cl- Iso-electric focussing (IEF) •Proteins separated in pH gradient formed by ampholytes •Where protein is at pH below pI, it is (+)-charged, and move towards anode •Where protein is at pH above pI, it is (-)-charged and moved towards cathode •At pH = pI, protein has no net charge, and is immobile •Proteins migrate to a pH region where they have no net charge •Proteins separated in terms of pI •Ampholytes poured into gel before setting •Buy pre-fabricated Immobilised pH gradient (IPG) strips •Wide range (pH3-10) or narrow range (pH8-9) strips 14 Two-dimensional gel electrophoresis (2DGE) Run IEF in the first dimension and SDS-PAGE in the second dimension 1st dimension IPG strip SDS-PAGE Resolved by mass 2nd dimension Resolved by pI Example of a 2D Gel 240µg of E. coli 2DGE, pH range 4 – 7, mass range 10 – 120kDa. 15 Resolution of 2DGE More proteins in proteome than can be resolved by standard 2DGE system Use 30cmX30cm gels (resolve 10 spots) Run multiple IEF strips at overlapping or adjacent pH ranges pH 3-12 pH 5-6 Use non-linear IPG stips, since most of the proteome has pI values between 4 and 7 Sensitivity of 2DGE •Proteins levels differ by 105-109 in a cell •Superabundant proteins are present at 1,000,000 copies per cell •Abundant proteins are present at 100,000 copies per cell •Rare at proteins are present at 1000 copies per cell •Thus, very rare proteins may not be visible on 2DGE gels •Very abundant proteins may obscure less abundant proteins •Solve sensitivity by increasing resolution •Narrow range IPGs •Prefractionation or affinity depletion 16 Spot-picker robot for 2DGE gels An LC/MS/MS system 17 Protein solubility Factors affecting protein solubility •Most proteins are highly soluble under physiological conditions (0.15-0.2M salt and neutral pH) •Solubility affected by: •Polar interactions with solvent molecules •Ionic interactions with the salt ions present •Repulsive electrostatic repulsions between •Like charges •Small, heterogenous aggregates + + + + + + - - - - + - - - + + + + + + + + + + + - + + + - + + - + + - + - + + + ++ + + - + + + + + + + δ- + + δ+ 18 Iso-electric precipitation pH < pI + + + + + + - + + - pH + + + + - •Repulsion + - + - - + - + + pH > pI pI - + - - + - + + - + - - - •Little repulsion •Hydrophobic interaction predominates •Also electrostatic attraction between opposite charges - - + + - •Repulsion •A protein is least soluble close to its pI “Salting in” proteins Attracting electrostatic charges + + + + aggregated + + + + + + - + + salt + -+ + + - + + + + - + + + + + + - - - + + - soluble - + -+ Ions screen electrostatic charge Attractive forces weaken 19 “Salting out” proteins - + + + - - Water molecules ordered next to exposed hydrophobic patches - + + salt - + + + + - + - - + - - + - - + Ions compete for water molecules Hydrophobic patches exposed Aggregation results - Hydrophobic association minimizes water molecule order Ring of ordered water molecules Hydrophobic droplet •Volume of sphere = 4πr3/3 •Surface of sphere = 4πr2 •1ml sphere has surface area of 4.8cm2 •2 spheres of 1ml will have combined area of 9.6mm2 •If the two droplets fuse, the combined volume is 2ml •The total surface area of the 2ml droplet is 7.7cm2 ∴if the two droplets fuse, the surface area will be less the number of water molecules that must be highly ordered is less the number of disordered water molecules increase increase in entropy 20 Increased temperature increases hydrophobic association temp More disordered water molecules •∆G = ∆H - T∆S •∆S is the change in entropy (order) •Change from order to disorder gives a positive ∆S, and ∴ a -∆G •Processes where ∆G < 0 (i.e., negative) occur spontaneously •At higher temperature, the disorder in the free water molecules increase ∴transfer of a water molecule from the ordered environment next to the hydrophobic droplet to the disordered state, will have a larger ∆S component •Thus, it becomes very favourable to fuse the droplets at higher temperatures Increased salt increases hydrophobic association ClNa+ salt ClCl- Na+ ClNa+ Na+ Cl- More disordered water molecules •At higher salt concentrations, weak interactions between the disordered water molecules are further disrupted, i.e., the molecules become even less ordered •transfer of a water molecule from the ordered environment next to the hydrophobic droplet to the disordered state, will have a larger ∆S component at higher salt •it becomes very favourable to fuse the droplets at higher salt concentrations 21 The Hofmeister series Salting out described by: log10(solubility) = A – m(salt concentration) A = constant that depends on pH and temperature m = independent of pH and temperature •Salt must not interact with protein (chaotropic) •Protein aggregation increases with increase in temperature •Anions with decreasing tendency to salt out lysozyme: •phosphate > sulphate > acetate > Cl- > Br- > nitrate > perchlorate •Cations with decreasing tendency to salt out lysozyme: • ammonium > K+ > Na+ • This series is known as the Hofmeister series Finding the best salting out protocol for your protein % Saturation First trial % Enzyme precipitated % Protein precipitated Purification factor 0-40 4 25 40-60 62 22 2.8 60-80 32 32 1.0 80 supernatant 2 21 Conclusion: Enzyme precipitated more in 40-60 than 60-80; try 45-70 Second trial 0-45 6 32 45-70 90 38 70 supernatant 4 30 2.4 Conclusion: Good recovery, but poorer purification; try 48-65 Third trial 0-48 10 35 48-65 75 25 65 supernatant 15 40 3.0 22 Effect of organic solvents on protein solubility •Organic solvents decrease dielectric constant of solvent •Displaces water molecules from and solvates hydrophobic patches - + + soluble + - - + - + organic solvent + + - + - - + - •Solvent displaced water molecules from hydrophobic patches •Increase in hydrophobic interaction •Also evidence of electrostatic attraction •Aggregation Solvents used to precipitate proteins •Ethanol •Acetone •Methanol •i-propanol •n-propanol •Dioxan •2-methoxyethanol •Precipitation must be performed below 10°C, otherwise proteins denature •Some organic polymers such as polyethylene glycol (PEG) acts like organic solvents, but are effective at much lower concentrations 23 Organic solvents and elevated temperatures Increasing temperature Internal hydrophobic interactions stabilizes protein •Elevated temperature causes increased molecular vibration •This allows organic solvent molecules to interact with internal hydrophobic regions •Internal hydrophobic interactions become disrupted Protein unfolds and denatures Gel filtration chromatography 24 Molecules are separated by size Sample of 3 different sized molecules (Stokes radius) Excluded Partially included Totally included Stationary phase Elution profile A280 Mobile phase Time Parameters of a gel filtration run Vo Ve Vt Ve > Vt ??? Vt = Vo + Vi For a column, Vo can be determined by eluting blue dextran (Mr = 2,000,000) Vi is internal volume plus particle volume (inaccessible to all), and is difficult to measure Vi can be calculated from Vt-Vo Excluded molecules elute at Vo Molecules that have access to full Vi elute at Vt What if elution volume Ve > Vt ? Inert stationary phase? 25 Partition coefficient The partition coefficient (Kd) for a molecule is given by Total volume accessible to molecule Ve - Vo Vt - Vo Total internal volume •If Kd = 0, the molecule is totally excluded •If Kd = 1, molecule has full access •If Kd > 1, there is adsorption to the column matrix •Kd is not the “true” partition coefficient, since Vi includes gel particle volume Resolution Ve1 Ve2 W2 W1 R=2 Ve2 – Ve1 W1 + W2 ∴The difference in elution volume divided by the average peak width, W If R = 1, we have baseline resolution 26 Theoretical plates •A chromatographic column can be thought of as a number of independent partition “cells” •Each such partition cell is known as a “theorical plate” •Column resolution depends on the number of theorical plates (N) on the column •number of theorical plates (N) is given by 5.55 Ve2 W2 •Where W is peak width, and Ve is the elution volume Height equivalent of a theoretical plate •The height equivalent of a theoretical plate (HETP) is given by the column length divided by the number of theoretical plates (N) • HETP = = L N LW2 5.55 Ve2 •Where L is the column height, W is peak width, and Ve is the elution volume 27 Van Deemter equation •The theoretical plate height is related to the elution speed of the mobile phase •H is a function of •A, diffusion of the molecule: the longer the molecule is on the column, the more it will diffuse •B, non-equilibrium, the longer partitioning between the stationary and mobile phase takes, the higher the plate height •C, column effects, including packing quality, particle shape and size distribution H= A + Bυ + C υ Finding the optimum flow rate van Deemter relation A (diffusion) B (non-equilibrium) C (column quality) A+B+C terms 5 Plate Height (H) 4 3 Optimum flow rate 2 1 0 0 5 10 15 20 Elution rate (ν) •A = 4, B = 0.2, C = 1 •As flow rate increases •Effect of diffusion decreases •Effect of non-equilibrium increases •Optimum flow rate (arrow) minimizes these two terms 28 Gel matrices Trade name Supplier Composition Comment Sephadex Pharmacia Cross-linked dextran Cannot be manufactured with exclusion limit > 600,000Da (particle not rigid, and collapses) Sephacryl Pharmacia N,N’methylenebisacrylamide cross-linked dextran Higher exclusion limits than Sephadex Polyacrylamide (Bio-Gel P) Bio-Rad N,N’methylenebisacrylamide cross-linked acrylamide Similar to Sephadex Sepharose Bio-Gel A Pharmacia Bio-Rad Agarose Very high exclusion limits Combined polyacrylamide-agarose gels Higher flow-rates than agarose Ultragel Gel matrix has optimum ~linear range Elution volume V0 Vi poor good poor Log10(Mr) 29 Ion-exchange chromatography Proteins have charges Ribbon Electrostatic surface 30 Charged (DEHKR) Acidic Aspartic acid, Asp, D negative charge Hydrophobicity = -3.0 MW 115Da pKa COOH = 2.19 pKa NH2 = 9.60 pI = 2.77 Glutamic acid, Glu, E negative charge Hydrophobicity = -2.6 MW 129Da pKa COOH = 2.19 pKa NH2 = 9.67 pI = 3.22 Basic Histidine, His, H Weak positive charge Hydrophobicity = -1.7 MW 137Da pKa COOH = 1.82 pKa NH2 = 9.17 pI = 7.59 Lysine, Lys, K positive charge Hydrophobicity = -4.6 MW 128Da pKa COOH = 2.18 pKa NH2 = 8.95 pI = 9.47 Arginine, Arg, R positive charge Hydrophobicity = -7.5 MW 156Da pKa COOH = 2.17 pKa NH2 = 9.04 pI = 10.76 The charge of protein depends on pKa 31 Principle of ion-exchange •IEX separates proteins on the basis of their net surface charge •An ion-exchange (IEX) resin is a resin than is negatively or positively charged at a given pH •Proteins with the opposite charge will bind to this resin •Cation exchanger binds positively charged proteins •Anion exchanger binds negative charged proteins •Electrostatic attraction between charged protein z1 and IEX resin charged group z2, is given by F= z1z2 Dr2 •The larger the (opposite) charges and the closer they are, the stronger the attraction •Ion exchange columns are typically eluted by increasing the ionic strength (salt concentration) of the buffer IEX bead matrices Resin matrices are composed of different materials •Cellulose •Agarose •Dextran •Synthetic polymers •Polyacrylamide IEX matrices will also act as gel filtration media 32 Functional groups +1 Q Anion exchangers DEAE 0 Cation exchangers CM S -1 3 4 5 6 7 8 9 10 pH •DEAE (diethylaminoethyl) and CM (carboxymethyl) are weak exchangers •Sulfonyl (S) and quaternary ammonium ions (Q) are strong exchangers Strong and weak exchangers Anion exchangers Strength Functional group Quaternary ammonium strong -O-CH2-N+-(CH3)3 Diethylaminoethyl weak -O-CH2-CH2-NH+-(CH2-CH3)2 Diethylaminopropyl weak O-CH2-CHOH-CH2-NH+-(CH2-CH3)2 Cation exchangers Functional group Sulfopropyl (S) strong O-CH2-CHOH-CH2-O-CH2-CH2-CH2-SO3- Methyl sulfonate (SP) strong O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-SO3- Carboxymethyl (CM) weak -O-CH2-COO- 33 A280 A280 A280 A280 v v v v charge Cation exchangers + 0 - A280 A280 A280 v v A280 pH Anion exchangers v v Affinity chromatography 34 Attachment of the ligand to the matrix The covalent attachment of the ligand to the matrix requires 1. Activation of the matrix 2. Attachment of the ligand to the activated group Many activated matrices are commercially available • • • • • CNBr-activated agarose Sepharose 4B 6-aminohexanoic acid (CH)-agarose 1,6-diaminohexane (AH) agarose Carbonyldiimidazole (CDI)-agarose Epoxy-agarose Attachment to CNBr-activated agarose OH gel OH CNBr O gel C NH O NH2R OH gel O C NHR NH2+ 35 Tagged recombinant proteins Tag Sequence Histidine HHHHHH C-myc EQKLISEEDL HA YPYDVPDYA VSV-G YTDIEMNRLGK HSV QPELAPEDPED V5 GKPIPNPLLGLDST FLAG DYKDDDDKG Glutathion-S-transferase 23kDa; 210 residues VSV-G: vesicular stomatitis virus G protein HSV: Herpes simplex virus Immobilized metal affinity chromatography (IMAC) •Based on the affinity of metal ion for basic amino acid residues •Ions mostly used include Ni2+, Co2+, Cu2+, Zn2+ and Fe2+ •Metal ion is immobilized by partial coordination binding by IDA, NTA and TED •Affinity is caused by metal ion not being fully coordinated •Solvent and buffer molecules coordinate weakly •Basic amino acid residues such as histidine, tryptophan and cysteine coordinate much more strongly •One can fuse a stretch of 6 contiguous histidine residues (6× his tag) to the C-terminal or N-terminal of a protein, and isolate the recombinant fusion protein on a Ni2+-agarose column • Protein is eluted with •imidazole (structural analog of histidine) •elution buffer pH can be dropped to 6, where histidine is protonated and cannot bind coordinately to Ni2+ 36 Metal chelators used in IMAC OH CH2CH CH2COOCH2 CH2 OH CH2CH N CH2 Imido diacetic acid (IDA) N CH COO- CH2COOCH2COO- Nitrilo triacetic acid (NTA) CH2COO- OH CH2CH CH2CH2N CH2 CH2COOCH2COO- N Tris(carboxymethyl) ethylene diamine (TED) CH2COO- Imidazole is structural analog of histidine C NH CH2 HC Histidine NH Imidazole 37 Tandem affinity purification (TAP) •Although one-step IMAC yield pure protein, it is seldom homogenous •TAP tag is based on having two tandem affinity tags in protein •Most frequent is calmodulin binding protein (CBP) followed by protein A •CBP binds to the protein calmodulin in the presence of Ca2+ •Protein A binds to the FC region of IgG immunoglobulins •The CBP and protein A sequences are separated by a TEV protease site (ENLYFQE) •This sequence is cleaved by TEV protease •The protein is first bound to Sepharose beads covered with IgG •Adsorbed protein cleaved with TEV protease and eluted •Binding to calmodulin column •Elution with Ca2+ •This allow two-step ultra-pure protein preparation •Very mild conditions allows isolation of protein complexes TAP tag purification scheme IgG sepharose column E. coli cell TAP-tagged protein Elute by TEV protease cleavage calmodulin column Ca2+ Ca2+ EDTA Pure protein Ca2+ EDTA Elute with EDTA or EGTA Ca2+ Ca2+ CBP binds to calmodulin only in presence of Ca2+ 38