Folding and Denaturation of Proteins PDF

Chapter 4:Folding and Denaturation (4.4) Chapter 3: Protein Purification (3.3-3.4) Objectives: Protein folding and denaturation Methods to characterize peptides and proteins 1 Common Questions About Peptides and Proteins What is its sequence...

Chapter 4:Folding and Denaturation (4.4) Chapter 3: Protein Purification (3.3-3.4) Objectives: Protein folding and denaturation Methods to characterize peptides and proteins 1 Common Questions About Peptides and Proteins What is its sequence and composition? What is its three-dimensional structure? How does it find its native fold? How does it achieve its biochemical role? How is its function regulated? How does it interact with other macromolecules? How is it related to other proteins? Where is it localized within the cell? 2 1  Protein Stability and Folding A protein’s function depends on its _________________ Loss of structural integrity with accompanying loss of activity is called __________ Proteins can be denatured by: heat or cold (affect vibrational and rotational energies) pH extremes (adjust protonation and deprotonation) 3 Protein Stability and Folding Cont’d Continued… Proteins can be denatured by: detergents – (hydrophobic tail & hydrophilic head) – break hydrophobic interior Eg: Triton X- 100 (nonionic) chaotropic agents: urea, guanidinium hydrochloride (disrupt stabilizing factors) Reducing agents: 2-mercaptoethanol 4 2  Protein Denaturation Thermal denaturation midpoint of the temperature range over which denaturation occurs is called the melting temperature, or Tm 5 Ribonuclease Refolding Experiment Ribonuclease is a small protein that contains eight cysteines linked via four _____________________ Urea in the presence of 2-mercaptoethanol ______________ ribonuclease. When urea and 2-mercaptoethanol are removed, the protein _____________________, and the correct disulfide bonds are reformed. The sequence alone determines the native conformation. The experiment is quite “simple” but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize. 6 3  Renaturation of unfolded, denatured ribonuclease Urea disrupts the hydrogen bonding, and mercaptoethanol (HOCH2CH2SH) cleaves the disulfide bonds = eight Cys residues Note: Folding dependent on primary structure 7 Protein Stability and Folding The precise nature that promote the folding of proteins have not been completely resolved Thermodynamically favorable process with overall __________ free energy change ΔG = ΔH - TΔS Negative free energy results in a balance of favorable and unfavorable enthalpy and entropy changes As polypeptide folds, favorable (-ΔH) with opposing factors, unfavorable decrease in entropy) as disorganized polypeptide folds into highly organized native state 8 4  Proteins Folding Follow a Distinct Path Protein folding is a ___________________ process Note: small regions of secondary structure are assembled first and then gradually incorporated into The numbers indicate the larger structures amino acid residues in this 56 residue peptide that have acquired their final structure Video:Sim ulation ofm illisecond protein folding https://www.youtube.com/watch?v=gFcp2Xpd29I 9 How Can Proteins Fold So Fast? Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast? It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox). Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable. ΔG = ΔH - TΔS Folding determined by thermodynamics 10 5  Thermodynamics Folding Free energy funnel native structure (N) at the bottom (lowest free-energy point) 11 Chaperones Prevent Misfolding and Aggregation of Unfolded Peptides Chaperones: assist unfolded proteins by protecting them from inappropriate interactions which can result in misfolding or aggregation Two major families: Hsp70 Chaperonins Note: Hsp70 binds to regions of unfolded polypeptides that are rich in hydrophobic residues. WHY? Hsp70 bound with unfolded polypeptides is delivered to chaperonins 12 6  Chaperonins Facilitate Folding A proposed pathway for the action of the E. coli chaperonins GroEL (a member of the Hsp60 protein family) and GroES. Each GroEL complex consists of 2 large chambers formed by 2 heptameric rings GroES, also a heptamer, blocks one of the GroEL chambers after an unfolded protein is Interior space where the bound inside protein are bound in the GroEL/GroES complex Folding occurs within the chamber, during the time it takes to hydrolyze the 7 ATP bound to the subunits in the heptameric ring GroES and the ADP molecules then dissociate, and the protein is released Protein misfolding is the molecular basis of a wide range of human diseases 13 Protein Misfolding Is the Basis of Numerous Human Diseases Native (correctly folded)  amyloid is a soluble globular protein, Misfolded  amyloid promotes aggregation at newly exposed protein-protein interface. Correctly folded helices are lost and peptides form  strands,  helices, and  sheets. 14 7  Chapter 4:Folding and Denaturation (4.4) Chapter 3: Protein Purification (3.3-3.4) Objectives: Protein folding and denaturation Methods to characterize peptides and proteins 15 Studying Proteins and Peptides Sometimes Requires Purification from a Mixture Polypeptides contain differing amino acid sequences. The sequence and arrangement of amino acids gives the polypeptide a chemical character (i.e., charged, polar, hydrophobic, etc.). Some polypeptides bind specific targets, which can be used to “fish them out” of a complex mixture. 16 8  A Mixture of Proteins Can Be Separated 17 Protein Purification - Features How a protein is purified will Protein source – tissue and from microbial cells depend on certain characteristics Cell Cell Extract Centrifuge Supernatant Chromatography Pure protein 18 9  Characteristics: Protein Solubility and pH At pI, the overall protein charge is ___zero______ At LOW pH, protein has a ____positive___ charge All are __protonated___ At HIGH pH, protein has a ____negative___ charge All are ____deprotonated___ Mathews , Van Holde, Ahern 2000, pg 49 Solubility vs pH 19 Protein Purification - Solubility Normally, water is forced to order around hydrophobic areas. In high salt, the salts will be solvated preferentially the ordered waters will be stripped off, exposing hydrophobic pockets proteins aggregate to bring hydrophobic pockets together. = H2O 20 10  (NH4)2SO4 Precipitation Decrease in the ordering of water will increase entropy (disorder) Isolates the desired protein, Makes precipitation favorable. then centrifuged at low-speed Proteins with larger and/or more solvent exposed hydrophobic Why low speed? patches will precipitate first. Proteins with less, will stay in solution longer. Solubility varies protein to protein, so it can be used to separate proteins 21 Protein Purification – Several Features Column Chromatography Column chromatography allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins. Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later. 22 11  Column Chromatography Fractionating proteins Stationary phase: solid support. resin Takes advantage of difference in or gel matrix eg: protein charge, size, binding silica affinity and others Mobile phase: Chromatographic methods enhanced buffered solution by use of Protein must also be HPLC - High Performance Liquid in the buffered Chromatography solution before added to the column FPLC – Fast Protein Liquid Chromatography After collection, They make use of high-pressure pump determine the protein and its amount in each NOTE: Protein will migrate faster or collection tube slower depending on its properties 23 Separation by Charge: Ion Exchange Separation is based on: charge at specific pH (away from its pI) salt concentration WHY is that so? NOTE: the beads does not interact with the protein 24 12  Question 25 Separation by Size: Size Exclusion Also known as____________ Separates protein according to _____________ Larger size comes out of the column________ Why? Matrix made up of porous beads, so _______ will enter those pore Insoluble in water but highly hydrated polymer eg: dextran, agarose 26 13  Separation by Binding: Affinity Separates protein by their _______________ Example of using this separation method: ATP-binding proteins Bound protein is eluted with ________ concentration of free ligand (causes competition between free ligand and ligand attached to the bead) High concentration of salt WHY? 27 Separation by Molecular Size Separates proteins from small solutes due to _________ Exchange of salt and buffer but not __________ Example: used to remove salts like ammonium sulfate Dialysis Kidney dialysis Procedure Stryer 7th ed 28 14  Question ralted to dialysis: Assuming you want to lower the high salt content of NaCl (100 mM) from your protein solution (10 mL) to almost no NaCl (~0 M). What would you do? 29 Electrophoresis for Protein Analysis Online image 30 15  Gel Electrophoresis Note: not typically used to purify protein. It is an analytical method to check the content of the protein mixture or to VERIFY purified protein It can affect the structure or function of the protein so typically used as an analytical method Online image Migration of __________ proteins in electric field 31 Components of the Polyacrylamide Gel Online image Acrylamide Makes the pore size of the gel 32 16  Gel Electrophoresis Sodium dodecyl sulfate (SDS) 33 SDS-PAGE Gel - Overview Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gel The method is used to check the protein content based on ________________ Lower concentration of acrylamide Hence the “natural” or intrinsic charge of the protein is not factored here SDS Hence, Sodium dodecyl sulfate (SDS) is a detergent is used to coat the protein with negative charge. WHY? Hint check the direction of the electric field ___________ will migrate faster Cathode region to anode region due to less resistance from the gel matrix 34 17  SDS PAGE Gel - Process 35 Protein Quality Visualized using a dye called Coomassie blue which binds to the proteins present in the gel. How does the dye bind to the protein and not the gel? Through ionic interactions between sulfonic acid groups on the dye and positive protein amine groups 36 18  Specific Activity Describes the Purity of the Protein of Interest Proteins in a complex mixture often require more than one purification to completely isolate the protein of interest. During purification, determination of the location of the protein of interest can be performed by tracking its behavior. If a protein has a specific function (e.g., binding insulin), the fraction that binds insulin best after each purification step will contain the most of the protein of interest. The function of the protein is called the “activity.” The ratio of activity to total protein concentration is called the “specific activity.” 37 Purification Table TABLE 3-5 Purification Table for a Hypothetical Enzyme What could affect activity? Fraction Total protein Specific activity Procedure or step Activity (units) volume (ml) (mg) (units/mg) 1. Crude cellular extract 1,400 10,000 100,000 10 2. Precipitation with ammonium sulfate 280 3,000 96,000 32 3. Ion-exchange chromatography 90 400 80,000 200 4. Size-exclusion chromatography 80 100 60,000 600 5. Affinity chromatography 6 3 45,000 15,000 Note: All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 95. After step 5, the enzyme in question has been purified by a factor of 1,500, as reflected in the increase in specific activity relative to that in the crude extract, and the yield of the enzyme is 45%, as reflected in the recovery of total activity. Activity: total units of enzymes in a flask Unit (U): 1.0 unit of enzyme activity is the amount of enzyme causing conversion of 1.0 µmol of substrate to product per minute at 25 ˚C Specificity activity: # of unit per mg of total of total proteins (units/mg). Specific Activity Describes the Purity of the Protein of Interest 38 19  Protein Sequencing It is essential to further biochemical analysis that we know the sequence of the protein we are studying. The actual sequence is generally determined from the DNA sequence. Edman degradation (classical method): – successive rounds of N-terminal modification, cleavage, and identification – can be used to identify protein with known sequence Mass spectrometry (modern method): – MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence – can be used to determine posttranslational modifications 39 Edman’s Degradation for Protein Sequencing Used to determine the amino acid sequence Steps: Step 3 1. React the peptide with a reagent (FDNB) which selectively label the terminal amino acid 2. Perform hydrolysis the protein using 6M HCl 3. Determine the amino acid by chromatography and comparison with standards 40 20  Protein Sequencing: Sanger Fragments 41 Proteases TABLE 3-6 The Specificity of Some Common Methods for Fragmenting Polypeptide Chains Reagent (biological source)a Cleavage pointsb Trypsin (bovine pancreas) Lys, Arg (C) Submaxillary protease (mouse submaxillary gland) Arg (C) Chymotrypsin (bovine pancreas) Phe, Trp, Tyr (C) Staphylococcus aureus V8 protease (bacterium S. aureus) Asp, Glu (C) Protease Asp-N-protease (bacterium Pseudomonas fragi) Asp, Glu (N) Pepsin (porcine stomach) Leu, Phe, Trp, Tyr (N) Endoproteinase Lys C (bacterium Lysobacter enzymogenes) Lys (C) Cyanogen bromide Met (C) aAll reagents except cyanogen bromide are proteases. All are available from commercial sources. bResidues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the carbonyl (C) or the amino (N) side of the indicated amino acid residues. 42 21  Protein Sequences as Clues to Evolutionary Relationships Sequences of proteins with identical functions from a wide range of species can be aligned and analyzed for differences. Differences indicate evolutionary divergences. Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth. 43 Consensus Sequences Necessary for function 44 22