Lecture 5. Tuesday, September 12. Protein Purification. Principles of Protein Structure, contd.

Lecture 5. Tuesday, September 12. Protein Purification. Principles of Protein Structure, contd.

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Unformatted text preview: Purification of Proteins Chem130/MCB100A Fall 2006 University of California, Berkeley Outline: • Proteins have different physical properties • These properties can be used to isolate populations of specific proteins from one another Source: Matthew Young Protein properties • • • • • Different solubilities Different lengths of amino acid chain Different charges Different 3D shapes Different surface features/ligand binding • Purifying proteins takes advantage of these differences to isolate a population of a protein of interest Protein Purification Flowchart cell Membranes, insoluble matter Soluble matter Darnell, Lodish, Baltimore, Molecular Cell Biology DNA, RNA, etc. A population of one protein of interest proteins 99% of all of the proteins in the cell Protein properties • Different solubilities • • • • Different lengths of amino acid chain Different charges Different 3D shapes Different surface features/ligand binding Releasing proteins from the cell • Physical methods – Shearing open the cells using pressure or strong sound waves • Chemical methods – Detergents, or enzymatic digestion • Centrifuge to separate insoluble membranes and proteins from soluble matter and proteins Protein properties • Different solubilities • Different lengths of amino acid chain • Different charges • Different 3D shapes • Different surface features/ligand binding Gel electrophoresis • Porous gel: – polyacrylamide • SDS (sodium dodecyl sulfate) – Detergent – Unfolds proteins – Gives all protein chains a net negative charge • Voltage – Top: (-) – Bottom: (+) • “run to the red” samples SDS-Page gel • Proteins travel at a rate inversely proportional to their size (number of residues) only – Shorter proteins travel down the gel faster • Proteins must be stained to see them Direction of travel (-) Size (KDa) (+) UV Absorbance λmax Trp Tyr Phe 280 274 257 Chromatography The beads are much bigger than proteins A column is packed with millions of tiny beads: Sample flow Protein properties • Different solubilities • Different lengths of amino acid chain • Different charges • Different 3D shapes • Different surface features/ligand binding • • Certain amino acids can act as acids and bases, with characteristic titration curves The amino acid sidechains have specific pKas: pH = -log[H+] = pKa + log[A-]/[AH] 00 75 50 25 0 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 Some characteristic pKas for amino acid sidechain groups pKa Arginine Lysine Aspartate Glutamate Cysteine Histidine 12.5 10.5 3.7 4.3 8.2 6.0 State at pH 7.0 (charge) protonated (+) protonated (+) deprot. (-) deprot. (-) protonated (0) deprot. (0) • At a given pH: – A functional group with a pKa greater than the buffer pH will be mostly protonated – A functional group with a pKa less than the buffer pH will be mostly deprotonated 100 75 50 25 0 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 • The pH of the solution and the pKa of the chemical group determine the protonation state of a specific side chain – At the pKa of a functional group, 50% of that group will be protonated – Note that the local environment of a functional group can alter it’s pKa Ion-Exchange chromatography Protein titration curve: Charge vs. pH Proteins interacting with charged beads • Releasing the proteins from the beads: • Change pH of buffer: – Change net charge on protein • Add salt: – Screen and attenuate the electrostatic attraction Ion Exchange [salt] UV Protein properties • Different solubilities • Different lengths of amino acid chain • Different charges • Different 3D shapes • Different surface features/ligand binding Size-exclusion chromatography Gel filtration beads are porous: The pores vary in size, but are large enough for proteins to enter them and become temporarily trapped Smaller proteins can enter more pores, and are hence trapped more often as they pass through the beads UV Abs. Time: Tube number: 8 9 10 11 12 13 14 15 16 17 18 Size (KDa) Protein properties • • • • Different solubilities Different lengths of amino acid chain Different charges Different 3D shapes • Different surface features/ ligand binding Affinity Chromatography • Releasing the protein: • Excess chemical ligand – Not bound to beads • Substance which binds the ligand better than the protein • Substance which disrupts the protein-ligand interaction • Examples of affinity interactions: – Metal binding proteins – ATP binding proteins – Drug binding proteins – Protein-protein interactions Final result Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY John Kuriyan: University of California, Berkeley Chem 130/MCB 100A, Fall 2006, Lecture 5 Review of elements of protein structure 1. In globular proteins, α helices and β sheets are often amphipathic (or amphiphilic –means the same thing). Normal α helix 3.6 residues per turn ⇒ 3.6 residues = 360° ⇒ 1 residue step = 100° helical wheel: residues are located every 100° 1 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY In a β sheet, alternate residues point up and down: β sheets are not straight, but rather are twisted: 2 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Simple patterns of secondary structural elements packing together are called motifs. e.g., β-α-β Motifs are assembled into domains Typical domains are 100-300 residues Longer proteins are assembled from multiple domains 3 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY If a domain is small, then all the secondary structural elements are likely to be amphipathic: In such a protein we don’t expect to find long uninterrupted stretches of hydrophobic sidechains. In a larger protein domain a secondary structural element may be entirely within the interior of the domain: For a folded protein structure, like this one, we could expect to find an uninterrupted stretch of hydrophobic amino acids. 4 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY How many hydrophobic amino acids would we expect to find in such a buried α-helix? How would this compare to the hydrophobic stretches in membrane proteins? Turn structures or loops are almost never found inside the membrane. Hence turn structures or loops are outside the membrane, where water can satisfy the hydrogen bonding requirement. 5 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY How big is a typical domain? Leucine (L, Leu) turns out to a “typical” amino acid in terms of mass. Total Mass = 113 (on average, mass/residue =110) So, ~100 residue domain has a mass of 100 x 113 = 11300 ≈ 11 kDa kiloDalton The interiors of proteins turn out to be very close packed, corresponding almost to perfect packing of spheres. Let us consider the packing of spheres of approximately the size of a methyl group: CH3 6 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Due to packing defects, assume that volume occupied by this sphere is (3.0)3 Å3 ≈ 27 Å3. Hence, the volume per Dalton is: SPECIFIC VOLUME 27 3 = 1⋅ 8 Å 15 ⇒ Volume of 11 kDa protein = 11 x 103 x 1⋅8 Å3 = 19⋅8 x 103 Å3 If we assume that the protein is a sphere, 7 Restricted: For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY What is the radius? V= 43 π r = 19 ⋅ 8 x 10 3 3 volume radius ⇒ r3 = 3 x 19 ⋅ 8 x 103 = 4726.9 4π ⇒ r = 15 Å This implies that the diameter of a 100 residue protein is ~33 Å, or more roughly ~30 Å. This is actually a very good estimate, with the diameter typically being close to ~30 Å. Covalent bonding often makes atoms pack more closely together than our assumption of 3.0 Å, which is an upper limit. Some packing defects compensate for this. 8 ...
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This note was uploaded on 01/12/2010 for the course MCB 100A taught by Professor Kuryian during the Fall '09 term at Berkeley.

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