CHMI 2227E Biochemistry I

1. CHMI 2227EBiochemistry I Protein purification and characterization CHMI 2227 - E.R. Gauthier, Ph.D.

2. Protein purification • Proteins are always found as part of complex mixtures; • While some proteins are quite abundant (antibodies in the blood), others are found in minute amounts (a few molecules per cell); • The first step to study the structure and/or function of a specific protein will always be its purification; • From a biological sample containing the protein of interest, a series of procedures are used sequentially to progressively obtain the protein in a « pure » form; • Protein purification is made much easier by previous knowledge of: • Basic characteristics of the protein: Mr, pI, solubility; • The availability of an assay to detect the presence of the protein (reagents, enzymatic reaction, physiological effect, etc). CHMI 2227 - E.R. Gauthier, Ph.D.

3. Crude protein extract Homogenization Coarse purification steps • Monitor presence of • protein of interest: • Activity • Purity • Quantity PURE! (well, let’s hope so…) Chromatography 1, 2, 3….x steps Protein purificationGeneral procedure CHMI 2227 - E.R. Gauthier, Ph.D.

4. Protein purification1. Coarse methods • Based on the solubility of the protein of interest under different conditions: • pH • Temperature • Ionic strength (i.e. salt concentration); • The idea is to use conditions that will lead to the precipitation of a lot of the proteins of the crude extract, while keeping the protein of interest in solution; • Allows one to get rid of « most of the junk » in the extract; CHMI 2227 - E.R. Gauthier, Ph.D.

5. Protein solubility is made possible by the interactions between the side chains and the solvent: If solvent is water: H bonds Electrostatic interactions If solvent is non-polar: Hydrophobic interactions; These interactions can be altered by changing the net charge of the protein, therefore by adjusting the pH of the solvent; As a general rule, proteins precipitate when the pH of the solvent equals their pI: Proteins aggregate and clump together. solubility pH pI Differential precipitation1.1. Precipitation by adjusting the pH CHMI 2227 - E.R. Gauthier, Ph.D.

6. Protein solubility can also be modified by altering the salt concentration in the extract; The added salts will take the place of the water around the protein; The salts will also neutralize the charges on the protein; These two events will favor protein aggregation and precipitation; Usually, salting out is performed in the presence of ammonium sulfate: (NH4)2SO4 More soluble in water than NaCl; Less amounts are required for protein precipitation. Proteins usually precipitate when a specific concentration of (NH4)2SO4 is reached; This allows one to clean up the cell extract in a stepwise fashion; The precipitated proteins can be discarded OR sujected to dialysis (to remove the salt) http://www.tulane.edu/~biochem/med/pure.htm Differential precipitation1.2.Salting out CHMI 2227 - E.R. Gauthier, Ph.D.

7. Differential precipitation1.2.Salting out PRECIPITATION!! CHMI 2227 - E.R. Gauthier, Ph.D.

8. Centrifugation: Separation by way of density; Dense (i.e. big) molecules will sediment (i.e. pellet) faster than light (i.e. small) molecules; Mixture of proteins in buffer: A: precipitates at salt [ ] = 15% B: precipitates at salt [ ] = 25 % C: precipitates at salt [ ] = 35% • Add (slowly) (NH4)2SO4 to 20% • Centrifuge to pellet precipitated proteins Pellet: protein A Supernatant: protein B + C • Add (slowly) (NH4)2SO4 to 30% • Centrifuge to pellet precipitated proteins Pellet: protein B Supernatant: protein C Differential precipitation1.2.Salting out CHMI 2227 - E.R. Gauthier, Ph.D.

9. Centrifugation • Widely used technique which uses centrifugal force to separate substances; • The centrifugal force applied varies depending on the substances to be separated: • 500 x g for whole cells • 15 000 x g for mitochondria • 150 000 x g for ribosomes Note: 1 x g = gravitational force on Earth at sea level (~ 9.8 m/s2) • Particulate substances will reach the bottom of the tube (i.e. they form a pellet) • The remaining liquid is called the supernate or supernatant liquid. • Can also be used to separate substances of different densities (denser substances will sediment faster than less dense substances) • e.g. separation of erythrocytes from leukocytes CHMI 2227 - E.R. Gauthier, Ph.D.

10. Because the salt used for the salting out generally inhibits the activity of proteins, it has to be removed; This is done by dialysis; The protein solution is simply put is a bag made of a semi-permeable membrane: Permeable to small molecules (e.g. salts); Not permeable to proteins; Also allows one to change buffer before performing the next step. Hours Dialysis CHMI 2227 - E.R. Gauthier, Ph.D.

11. After using crude methods to concentrate our protein of interest, other, more refined techniques must be used to get rid of other proteins; Usually achieved by different types of chromatography: Ion exchange; Molecular sieve; Affinity Usually performed by Fast Protein Liquid Chromatography (FPLC) Buffer reservoir Column Sample injector Fraction collector Pumps Protein purification2. Fine methods CHMI 2227 - E.R. Gauthier, Ph.D.

12. Allows proteins to be separated according to their charge; Protein mixture is placed in a buffer, the pH of which will give a specific, net charge to the protein of interest; The mixture is then loaded onto a chromatographic column containing the appropriate separation medium; Cation exchange: Phosphocellulose Carboxymethyl (CM) cellulose Anion exchange: Diethylaminoethyl (DEAE) cellulose Protein purification2.1. Ion exchange chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

13. Although a change of buffer (with a different pH) could succeed in eluting the proteins attached to the column, this is rarely done since a change in pH could denature the protein or worse, lead to its precipitation; Elution is usually performed by adding a buffer with a salt (NaCl) concentration that will prevent protein binding to the column; Elution can be performed using linear or stepwise gradients of salt concentration; Adsorption Desorption NaCl Anion exchanger Example of cation exchange chromatography Protein purification2.1. Ion exchange chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

14. Progressive, linear change in NaCl concentration Add buffer Stepwise gradient of NaCl concentration Protein purification2.1. Ion exchange chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

15. Also called gel filtration chromatography or size exclusion chromatography; Uses beads that contain pores of a specific size: Proteins larger than the pores cannot enter the beads, have less volume to go through and elute first; Proteins smaller than the pores will enter the beads and their elution will be delayed: they will elute later; Proteins are therefore separated according to their molecular mass: The availability of different types of beads with different ranges of pore size allows one to select the right chromatographic media for their separation requirements; Protein purification2.2. Molecular sieve chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

16. CHMI 2227 - E.R. Gauthier, Ph.D. http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_Prod~SelGuides~Media~GFSelG

17. Gel filtration has many applications: 1) Protein purification 2) Desalting NOT the same thing as salting out; The pores are so small than all the proteins go through and only the elution of salts is delayed; 3) Molecular mass determination: Requires one to run a set of standards first (a mixture of proteins of known Mr); Allows one to determine whether his/her favorite protein is part of a multimeric complex. WHY? Protein purification2.2. Molecular sieve chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

18. In this type of chromatography, beads are linked to a molecule called a ligand, which can only bind the protein of interest; Ligand: antibody, substrate, metals, other protein/macromolecule interacting with the protein of interest. When a mixture of partially purified proteins is filtered through this type of column, only the protein of interest is bound. The other, contaminating proteins are washed out; The protein of interest is then eluted by the addition of an excess of unbound ligand; Very powerful method but: Media is expensive (cannot perform large scale purifications) Limited availability of ligand-bonded beads; Requires prior knowledge of a ligand that can bind the protein of interest (which is seldom the case). Protein purification2.3. Affinity chromatography CHMI 2227 - E.R. Gauthier, Ph.D.

19. Analysis of proteinsElectrophoresis • During the purification process, the presence and purity of the protein of interest needs to be assessed; • Also, methods are required to study proteins without the need to purify them; • The most popular and simple methods for analyzing rely on the principle of electrophoresis; • Electrophoresis involves the separation of proteins via their migration through a gel, under an electric field. • The material generally used to separate proteins by electrophoresis is polyacrylamide. CHMI 2227 - E.R. Gauthier, Ph.D.

20. SDS Electrophoresis1. SDS-PAGE Electrophoresis • In SDS-PAGE, proteins are first placed in a buffer containing the detergent sodium dodecyl sulphate (SDS); • SDS will cover each protein similarly: approx 1 SDS molecule for every 2 amino acids; • This has the consequence of giving each protein the same density of negative charges, independent of their pI. • b-mercaptoethanol (HS-CH2-CH2-OH) is also frequently (but NOT always) added to break disulfide bonds. • Therefore, the only variable that can modify the migration of proteins in a gel will be their size; CHMI 2227 - E.R. Gauthier, Ph.D.

21. Electrophoresis1. SDS-PAGE Electrophoresis • The proteins are then placed in the wells of a SDS-PAGE gel, and subjected to an electric field; • The proteins then migrate towards the positively-charged anode: the distance they will migrate depends on their size; • Proteins are then stained in the gel, generally with a blue dye called Coomassie Blue; • By placing in an adjacent well a mixture of proteins of known molecular mass (protein standards), one can determine the molecular mass of the protein of interest. • Of note: treatment with SDS and b-ME denatures the proteins and breaks all interactions with other proteins: the protein will migrate as if it were a single polypeptide. • Sometimes, NATIVE PAGE is performed: in this situation, no SDS or b-ME is added. This allows proteins part of multimeric complexes to be studied. WHY? CHMI 2227 - E.R. Gauthier, Ph.D.

22. Electrophoresis1. SDS-PAGE Electrophoresis www.biology.ucsd.edu/classes/bggn224.FA06/crash_2.ppt http://wine1.sb.fsu.edu/bch5425/lect20/lect20.htm CHMI 2227 - E.R. Gauthier, Ph.D.

23. Electrophoresis1. SDS-PAGE Electrophoresis CHMI 2227 - E.R. Gauthier, Ph.D.

24. Log Mr Distance migrated from well (cm) Electrophoresis1. SDS-PAGE Electrophoresis CHMI 2227 - E.R. Gauthier, Ph.D.

25. Electrophoresis2. Isoelectrofocusing • Here, proteins are separated according to their pI; • The mixture of proteins is placed in the wells of an acrylamide gel containing a gradient of pH along its length; • Upon subjecting the proteins to an electric field, they will migrate in the gel according to their charge: • Positively charged proteins migrate towards the negative cathode; • Negatively charged proteins migrate towards the positive anode; • When a protein reaches a zone in the gel where the pH = pI, it carries no net charges and stops migrating; • This allows one to determine the pI of the protein of interest; CHMI 2227 - E.R. Gauthier, Ph.D.

26. pH in gel = pI of proteins Electrophoresis2. Isoelectrofocusing CHMI 2227 - E.R. Gauthier, Ph.D.

27. Here, the proteins are FIRST separated according to their pI by IEF. THEN, the strip of IEF gel is placed on top of a PAGE-SDS gel: the proteins will then be separated according to their Mr; Allows a much higher resolution of the proteins present in the mixture of interest; Electrophoresis3. Two-dimensional gel electrophoresis CHMI 2227 - E.R. Gauthier, Ph.D.

28. Each spot is a single protein Electrophoresis3. Two-dimensional gel electrophoresis CHMI 2227 - E.R. Gauthier, Ph.D.

29. This is a powerful application of SDS-PAGE; Allows the detection of a single protein of interest present in a complex mixture of proteins; Makes use of the properties of antibodies to specifically bind a unique molecule with high affinity; Antibodies: 4 chains: 2 light chains (25 kDa ea) and 2 heavy chains (50 kDa each); Each antibody possesses two identical binding site for an antigen (antigen: whatever the antibody specifically and uniquely binds to) Structure of an antibody Electrophoresis4. Western blot analysis CHMI 2227 - E.R. Gauthier, Ph.D.

30. Electrophoresis4. Western blot analysis CHMI 2227 - E.R. Gauthier, Ph.D.

31. Protein purificationExample and data analysis • At each step, took 3 samples: • One for protein quantification • One for protein activity (assay) • One for PAGE-SDS • The rest was subjected to the next purification step; CHMI 2227 - E.R. Gauthier, Ph.D.

32. Coomassie-blue-stained PAGE-SDS gel Protein purificationExample and data analysis CHMI 2227 - E.R. Gauthier, Ph.D.

33. Protein purificationExample and data analysis • Important values to obtain to get an estimation of the success of the purification (especially when setting up the procedure): • Specific activity (units/mg): Total activity (U)/ Total protein (mg) • Yield: (Total activity at Step Y / Total activity in crude extract) x 100; • Purification level: Specific activity at Step Y / Specific activity in crude extract; CHMI 2227 - E.R. Gauthier, Ph.D.

34. Protein sequencing • Every functional and structural properties depend on the order of amino acids in the polypeptides (their sequence); • The 3-D structure of a protein is uniquely dictated by its amino acid sequence; • Several genetically-heritable diseases are caused by a change (insertion, deletion, substitution) of one or more amino acids in the sequence of one protein (e.g. DY508 in cystic fibrosis); • Amino acids important in the structure/function of a protein will not change rapidly during evolution. • Comparisions between the amino acid sequences of proteins from different species can reveal unknown functions/properties of the protein of interest. CHMI 2227 - E.R. Gauthier, Ph.D.

35. Hemoglobin is a tetramer made of 2 copies each of 2 polypeptides: HbA and HbB Sickle cell anemia is caused by a single, heritable mutation in HbB: Glu replaced by Val Creates a hydrophobic patch (why?) that causes the aggregation of the mutated hemoglobin (called HbS). This aggregated (i,e, clumped) HbS forms long fibers that change the shape of the erythrocytes. These elongated red blood cells hinder blood flow. These elongated blood cells are also very fragile and burst easily (hence the anemia). BUT: because the malaria parasite grows in erythrocytes, people with sickle cell anemia are more resistant to malaria. Normal red blood cell Sickled red blood cell Protein sequence - Sickle-cell anemia CHMI 2227 - E.R. Gauthier, Ph.D.

36. Determination of protein sequence1. Enzyme mapping • Based on the property of some enzymes to cut the peptide bonds next to specific amino acids; NOTE: Trypsin, Chymotrypsin, protease V8, pepsin and thermolysin do NOT cut if Pro is part of the peptide bond. CHMI 2227 - E.R. Gauthier, Ph.D.

37. Determination of protein sequence1. Enzyme mapping – example 1 CHMI 2227 - E.R. Gauthier, Ph.D.

38. HCl 6M: Ala, Gly2, Lys, Met, Ser, Thr, Tyr CNBr: 2 peptides were obtained: Peptide 1: Ala, Gly, Lys, Thr Peptide 2: Gly, Met, Ser, Tyr Trypsin: 2 peptides were obtained: Peptide 3: Ala, Gly Peptide 4: Gly, Lys, Met, Ser, Thr, Tyr Chymotrypsin: 2 peptides were obtained: Peptide 5: Gly, Tyr Peptide 6: Ala, Gly, Lys, Met, Ser, Thr FDNB: yields Gly Carboxypeptidase A: yields Gly Determination of protein sequence1. Enzyme mapping – example 2 • The following data were obtained after treating an octopeptide with the following reagents: • What is the sequence of this peptide? CHMI 2227 - E.R. Gauthier, Ph.D.

39. Based on the use of phenyl isothiocyanate (aka PTC; Edman’s reagent); PTC reacts with and labels the amino acid at the N-terminus of the peptide; The PTC-labeled amino acid is released from the peptide and identified by chromatography; Cycles of labeling and release allow one to determine the sequence of the peptide. Determination of protein sequence2. Edman degradation CHMI 2227 - E.R. Gauthier, Ph.D.

40. Determination of protein sequence2. Edman degradation Identify CHMI 2227 - E.R. Gauthier, Ph.D.

41. Determination of protein sequence3. Mass spectrometry • Very powerful, now trendy technique to identify and sequence proteins; • Proteins are vaporised into ionized fragments with the use of a laser; • Even proteins cut out of a SDS-PAGE gel can be use!!! • The fragments are separated and their molecular mass determined; • From the molecular mass of the fragments, the peptide can be identified; • In tandem MS (MS-MS), fragments obtained after one MS run are subjected to a second fragmentation into even smaller fragments: the mass of these smaller fragments can be used to determine the amino acid sequence of the fragment. CHMI 2227 - E.R. Gauthier, Ph.D.

42. Determination of protein sequence3. Mass spectrometry CHMI 2227 - E.R. Gauthier, Ph.D.

43. Determination of protein sequence3. Mass spectrometry CHMI 2227 - E.R. Gauthier, Ph.D.

44. Example of protein purificationApoptosis: a form of cell death Normal Autophagy Current Opinion in Cell Biology 2004, 16:663–669 Apoptosis Necrosis CHMI 2227 - E.R. Gauthier, Ph.D.

45. Apoptosis – morphological aspects Shrinkage http://www.nature.com/nrm/journal/v9/n3/extref/nrm2312-s1.mov Blebbing Fragmentation

46. Apoptosis – morphological aspects http://www.nature.com/nrm/journal/v9/n3/extref/nrm2312-s1.mov

47. Apoptosis and physiology http://www.wikiwak.com/image/Celldeath.jpg

48. Example of protein purificationAcinus: a protein involved in cell death NATURE |VOL 401 | 9 SEPTEMBER 1999 CHMI 2227 - E.R. Gauthier, Ph.D.

49. Chromatin condensation and nuclear fragmentation duringapoptosis Nature Reviews| molecular cell biology volume9 | march2008 | 231

50. Example of protein purificationAcinus: a protein involved in cell death CHMI 2227 - E.R. Gauthier, Ph.D.