5 1 MCB 450 Lecture 5 Techniques in Protein Biochemistry Tissue Cell Fractionation Centrifugation Protein Purification Chromatography Electrophoresis Antibodies Peptide fragmentation sequencing Edman Degradation Analysis of Protein Structure by X ray Crystallography 5 2 Why purify proteins Study their enzymatic activity and what factors affect activity Determine their structures e g by X ray diffraction so we can relate structure to function Find compounds that block enzyme activity medicines Raise antibodies to them so we can detect the protein in living cells Use enzymes therapeutically industrially Challenge Need to get our protein out of the tissues cells that contain it and separate it from all other proteins 5 3 Tissue cell disruption Shear cells between close fitting plunger wall of thick tube Tissue homogenate cell suspension Force cells through small hole using high pressure Grind in mortar pestle w sand alumina liq N2 http cellbiologyolm stevegallik org node 74 Bacterial cells Sonication break cells w high frequency sound 5 4 Different cell components can be separated by centrifugation Differential centrifugation Spin at successively higher g forces faster speeds Exploits the fact that different cellular components have different densities sediment pellet at different g forces 5 5 Density gradient centrifugation Separates protein complexes and different cellular membranes e g sucrose solutions LESS DENSE MORE DENSE During centrifugation at high g component migrates down tube to region of equal density 5 6 Density gradient centrifugation Collection of different fractions LESS DENSE MORE DENSE 5 7 What does S mean Svedberg Units Sedimentation Coefficient S depends on particle shape size density mass density of the solution The smaller the S the more s l o w l y the particle molecule moves in a centrifugal field The functional ribosome is 70S It is composed of one 30S subunit and one 50S subunit 5 8 Protein purification Goal Separate your protein from all others in a cell extract Strategy Use several different procedures exact procedures order in which they are carried out differ from protein to protein Procedures exploit characteristic properties of the protein Protein property Method Solubility Increasing ionic strength at first increases solubility of proteins salting in then decreases it salting out Size shape Dialysis size exclusion chromatography gel filtration Charge pI Ion exchange chromatography electrophoresis Binding to small molecules Affinity chromatography small molecule immobilized For most protein purifications the steps are carried out at 4 C to slow down degradation crude samples contain proteases 5 9 Differences in protein solubility as a function of salt concentration Increasing ionic strength at first increases protein solubility At high salt concs competition between added salt ions and dissolved protein for water molecules Hydration shell is stripped off protein and it precipitates 5 10 Separation by size dialysis Can use dialysis to remove excess salt from a protein solution or to change the buffer a protein is in 5 11 Protein purification by chromatography Principle of chromatography Separation of molecules based on their relative mobilities through a matrix Separation of molecules is increased by retarding their relative mobilities by increasing their interactions with the matrix Examples Gel filtration mobility differences due to size Ion exchange mobility differences due to differences in electrostatic attraction to matrix Affinity chromatography immobilization due to binding to a specific ligand 5 12 Separation by size gel filtration chromatogaphy A buffer the protein can dissolve in collect fractions Liquid chromatography in a column is the most widely used method for separating proteins 5 13 Beads for gel filtration Often high mol wt polymers of glucose Sephadex Pore size in beads can be varied Exclude proteins above a certain mol wt which then pass down column more quickly Can calibrate column with standard proteins of known mol wt estimate size of unknown 5 14 Separation by charge ion exchange chromatography Compare pI of protein to the solvent pH RELATIVELY UN PROTONATED if pH pI protein will be vely charged if pH pI protein will be vely charged RELATIVELY PROTONATED Pass protein mixture through a column of ion exchanger resin polymer containing bound charged groups Cation exchanger pKa groups binds vely charged proteins Low Anion exchanger High pKa group binds vely charged proteins 5 15 CATION EXCHANGERS MOST EFFECTIVE WHEN pKa resin pH pI protein So resin vely charged So protein vely charged deprotonated protonated 5 16 ANION EXCHANGERS MOST EFFECTIVE WHEN pI protein pH pKa resin So protein vely charged So resin vely charged deprotonated protonated 5 17 Ion exchange chromatography on a cation exchanger pH of buffer pI of protein so protein vely charged pH of buffer pKa of resin so resin vely charged 5 18 Getting proteins off the cation exchanger Change the pH In this case raising pH will remove ve charge from the protein increase the ionic strength Na ions will compete with vely charged groups on a protein for binding to a cation exchanger 5 19 Separation by binding specificity affinity chromatography Exploits the fact that some proteins bind very tightly to a specific ligand Immobilize the ligand on a column 5 20 Displaying proteins by SDS polyacrylamide gel electrophoresis SDS PAGE 1 Binds to most proteins in amounts proportional to mol wt of the protein 1 SDS 2 Bound SDS contributes large ve charge intrinsic charge of protein so proteins are separated almost exclusively on the basis of mol wt Protein samples are denatured by heating in SDS mercaptoethanol to reduce S S bonds denaturing electrophoresis 5 21 Displaying proteins by SDS polyacrylamide gel electrophoresis SDS PAGE 2 Samples of denatured proteins loaded at top of polyacrylamide gel held between two glass plates POLYACRYLAMIDE ACTS AS AS A MOLECULAR SIEVE 5 22 Displaying proteins by SDS polyacrylamide gel electrophoresis SDS PAGE 3 Before After Mixture of SDSmercapto denatured proteins 5 23 Proteins separated by SDS PAGE can be visualized by staining gel with a dye such as Coomassie Blue Standards 200 kDa 150 kDa 100 kDa 75 kDa 60 kDa 35 kDa 25 kDa 12 kDa 6 5 kDa 5 24 Isoelectric focusing to separate proteins according to pI in gel containing a stationary pH gradient NO SDS LOW pH in GEL ve electrode anode HIGH pH IN GEL ve electrode cathode In the high pH region proteins