Exam 1 Recitation 2024 PDF

The Amino Acid Residues in Proteins are L Stereoisomers two possible stereoisomers = enantiomers optically active D, L system specifies absolute configuration  Answer Which amino acid has three pKa values? E. cysteine C...

The Amino Acid Residues in Proteins are L Stereoisomers two possible stereoisomers = enantiomers optically active D, L system specifies absolute configuration  Answer Which amino acid has three pKa values? E. cysteine Cysteine has three pKa values: pK1 (—COOH): 1.96 pK2 (—NH3+): 10.28 pK3 (R group): 8.18 Nonpolar, aliphatic R groups the hydrophobic effect stabilizes protein structure Aromatic R Groups R groups absorb UV light at 270–280 nm can contribute to the hydrophobic effect Polar, Uncharged R Groups R groups can form hydrogen bonds cysteine can form disulfide bonds Positively Charged R Groups have significant positive charge at pH 7.0 Negatively Charged R Groups have a net negative charge at pH 7.0 Amino Acids Can Act as Acids or Bases amino groups, carboxyl groups, and ionizable R groups = weak acids and bases zwitterion occurs at neutral pH  Isoelectric Point, pI for amino acids without ionizable side chains, the isoelectric point (pI) is: pK1 + pK 2 pI = 2 pH = pI = net charge is zero (amino acid least soluble in water, does not migrate in electric field) pH > pI = net negative change pH < pI = net positive charge  Peptides Are Chains of Amino Acids peptide bond: – covalent – formed through condensation – broken through hydrolysis  Answer Which statement is correct about peptides? B. Peptides have their amino acid sequences written from the N-terminus. Peptides are named beginning with the amino- terminal (N-terminal) residue.  Estimating the Number of Amino Acid Residues number of residues = molecular weight/110 average molecular weight of amino acid = ~128 molecule of water removed to form peptide bond = 18 128 – 18 = 110  Some Proteins Contain Chemical Groups Other Than Amino Acids conjugated proteins = Table 3-4 Conjugated Proteins contain permanently Class Prosthetic group Example associated chemical Lipoproteins Lipids 1-Lipoprotein of blood components (Fig. 17-2) – non–amino acid part Glycoproteins Carbohydrates Immunoglobulin G (Fig. 5-20) = prosthetic group Phosphoproteins Phosphate groups Glycogen phosphorylase (Fig. 6-39) lipoproteins contain Hemoproteins Heme (iron porphyrin) Hemoglobin (Figs 5-8 to 5-11) lipids Flavoproteins Flavin nucleotides Succinate dehydrogenase (Fig. 19-9) glycoproteins contain Metallproteins Iron Zinc Ferritin (Box 16-1) Alcohol dehrogenase sugars (Fig. 14-12) Calcium Calmodulin (Fig. 12-17) Molybdenum Dinitrogenase (Fig. 22-3) metalloproteins contain Copper Complex IV (Fig. 19-12) specific metals  Proteins Can Be Separated and Purified separated based on: – size – charge – binding properties – protein solubility  Column Chromatography first step = buffered solution (mobile phase) migrates through porous solid material (solid phase) second step = buffered solution containing protein migrates through solid phase protein properties affect migration rates  Answer Which component is absolutely necessary for the purification of a protein? C. a means of detecting the protein To study a protein in detail, researchers must be able to separate it from other proteins in pure form and must have the techniques to determine its properties. Ion-Exchange Chromatography separates based on sign and magnitude of the net electric charge pH and concentration of free salt ions affect protein affinity uses bound charged groups: – cation exchangers – anion exchangers Size-Exclusion Chromatography also called gel filtration chromatography separates based on size large proteins emerge from the column before small proteins do  Affinity Chromatography separates based on binding affinity eluted by high concentration of salt or ligand  Answer Which protein would elute first from a gel filtration column? C. protein C, a homodimer with protomer Mr = 35,300 Size-exclusion chromatography, also called gel filtration, separates proteins according to size. In this method, large proteins emerge from the column sooner than small ones do.  Sequential Purification Steps Decrease Sample Size Table 3-5 A Hypothetical Purification Table for an Enzyme Fraction Specific volume Total Activity activity Procedure or step (mL) protein (mg) (units) (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 chromatography80 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 90. final specific activity: starting specific activity ratio = purification factor percentage of the final activity/starting activity = percent yield  Proteins Can Be Separated and Characterized by Electrophoresis electrophoresis = visualize and characterize purified proteins can be used to estimate: – number of different proteins in a mixture – degree of purity – isoelectric point – approximate molecular weight Electrophoresis for Protein Analysis uses cross-linked polymer polyacrylamide gels proteins migrate based on charge-to- mass ratio visualization = Coomassie blue dye binds to proteins https://www.youtube.com/watch?v=MILiO1XnuqQ  Levels of Structure in Proteins four levels: – primary structure = covalent bonds linking amino acid residues in a polypeptide chain – secondary structure = recurring structural patterns – tertiary structure = 3D folding of polypeptide – quaternary structure = 2+ polypeptide subunits A Protein’s Conformation Is Stabilized Largely by Weak Interactions stability = tendency of a protein to maintain a native conformation unfolded proteins have high conformational entropy chemical interactions stabilize native conformations: – strong disulfide (covalent) bonds are uncommon – weak (noncovalent) interactions and forces are numerous hydrogen bonds hydrophobic effect ionic interactions Van der Waals interaction Packing of Hydrophobic Amino Acids Away from Water Favors Protein Folding hydrophobic effect solvation layer = highly structured shell of H2O around a hydrophobic molecule – decreases when nonpolar groups cluster together – decrease causes a favorable increase in net entropy hydrophobic R chains form a hydrophobic protein core  Answer Which item is the predominant factor in protein stability? B. the hydrophobic effect The hydrophobic effect, derived from the increase in entropy of the surrounding water when nonpolar molecules or groups are clustered together, makes the major contribution to stabilizing the globular form of most soluble proteins.  The Peptide Bond Is Rigid and Planar 3 covalent bonds separate the α carbons of adjacent amino acid residues: Cα —C—N—Cα resonance between the carbonyl oxygen and the amide nitrogen partial negative charge and partial positive charge sets up a small electric dipole  Peptide C—N Bonds Cannot Rotate Freely 6 atoms of the peptide group lie in a single plane partial double-bond character of C—N peptide bond prevents rotation, limiting range of conformations  The α Helix Is a Common Protein Secondary Structure α helix = simplest arrangement, maximum number of hydrogen bonds – backbone wound around an imaginary longitudinal axis – R groups protrude out from the backbone – each helical turn = 3.6 residues, 5.4 Å  Intrahelical Hydrogen Bonds between hydrogen atom attached to the electronegative nitrogen atom of residue n and the electronegative n+4 carbonyl oxygen atom of residue n + 4 n confers significant stability https://www.youtube.com/watch?v=PeFdl6KmxYM  Proline and Glycine Occur Infrequently in an α Helix proline = introduces destabilizing kink in helix – nitrogen atom is part of rigid ring – rotation about N—Cα bond not possible glycine = high conformational flexibility, take up coiled structures  Adjacent Polypeptide Chains in a β Sheet Can Be Antiparallel or Parallel antiparallel = opposite orientation – occur more frequently parallel = same orientation H bonds form between backbone atoms of adjacent segments https://www.youtube.com/watch?v=jT1XvChhJ8Y  Common Secondary Structures Have Characteristic Dihedral Angles dihedral angles (phi) and (psi) associated with each residue completely described secondary structure Ramachandran plots: – visualize all and angles – test quality of three-dimensional protein structures Secondary Structure Conformations are Defined by and Values  The Structure of Collagen collagen = found in connective tissue – secondary structure = left-handed, repeating tripeptide unit Gly–X–Y, where X is often Pro and Y is often 4-Hyp – tertiary and quaternary structure = right-handed twisting of 3 separate polypeptides Scurvy, Vitamin C, and Collagen Formation scurvy is caused by a lack of vitamin C – characterized by general degeneration of connective tissue vitamin C is required for the hydroxylation of proline and lysine in collagen Some Proteins or Protein Segments Are Intrinsically Disordered intrinsically disordered proteins: – lack definable structure – often lack a hydrophobic core – high densities of charged residues (Lys, Arg, Glu) and Pro – facilitates a protein to interact with multiple binding partners Loss of Protein Structure Results in Loss of Function denaturation = loss of three-dimensional structure sufficient to cause loss of function – can occur by heat, pH extremes, miscible organic solvents, certain solutes, detergents – often leads to protein precipitation Amino Acid Sequence Determines Tertiary Structure renaturation = process by which certain denatured globular proteins regain their native structure and biological activity Anfinsen experiment showed the amino acid sequence contains all the information required to fold the chain  Answer Denaturing followed by renaturing of a protein: B. demonstrates that primary structure dictates tertiary structure. The Anfinsen experiment provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, three-dimensional structure.  Polypeptides Fold Rapidly by a Stepwise Process local secondary structures fold first – ionic interactions play an important role longer range interactions follow – hydrophobic effect plays a significant role process continues until the entire polypeptide folds https://www.youtube.com/watch?v=gFcp2Xpd29I  Defects in Protein Folding Are the Molecular Basis for Many Human Genetic Disorders amyloid fiber = protein secreted in a misfolded state and converted to an insoluble extracellular fiber amyloidose diseases: type 2 diabetes, Alzheimer disease, Huntington disease, and Parkinson disease amyloid polypeptide (IAPP or amylin) deposits in islet cells Gradually less and less insulin created type 2 diabetes  Neurodegenerative Conditions Alzheimer disease = associated with extracellular amyloid deposition by neurons, involving the amyloid- peptide Parkinson disease = misfolded form -synuclein aggregates into spherical filamentous masses called Lewy bodies Huntington disease = involves the intracellular aggregation of huntingtin, a protein with long polyglutamine repeat  Oxygen Can Bind to a Heme Prosthetic Group oxygen: – poorly soluble in aqueous solutions – diffusion through tissues is ineffective over large distances – transition metals have strong tendency to bind (iron, copper)  Heme Prosthetic Group heme = protein- bound prosthetic group – present in myoglobin and hemoglobin – consists of a complex organic ring structure, protoporphyrin, with a bound Fe2+ atom  Coordination Bonds of Iron six coordination bonds: – four to nitrogen atoms in the flat porphyrin ring – two perpendicular to the porphyrin  Perpendicular Coordination Bonds two perpendicular coordination bonds: – one is occupied by a side-chain nitrogen of a highly conserved proximal His residue – one is the binding site for molecular oxygen (O2) Fe2+ binds O2 reversibly Fe3+ does not bind O2 Tetrahedral bipyramid  Answer The heme prosthetic group: A. consists of protoporphyrin and an iron (II) ion. Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2+) state.  Graphical Representations of Ligand Binding binding sites occupied [PL] Y= = total binding sites [PL] + [P] [L] [L] Y= = [L] + 1 [L] +K Ka [L] at which ½ of the available ligand- binding sites are occupied (Y = 0.5) corresponds to Kd Ka: association constant Kd: dissociation constant, Kd=1/Ka Ka [PL] + Ka= [P] [L] Kd  Answer Protein B has higher affinity. Larger Ka, Smaller Kd higher affinity Hemoglobin Undergoes a Structural Change on Binding Oxygen two conformations of Greater number hemoglobin: of ion pairs – R state = O2 has a higher affinity for hemoglobin – T state = more stable when O2 is absent, predominant conformation of deoxyhemoglobin  The T R Transition R state = O2 has a higher affinity for hemoglobin T state = more stable when O2 is absent O2 binding to hemoglobin in the T state triggers a conformational change to the R state – subunit pairs slide past each other and rotate – the pocket between the subunits narrow – some ion pairs that stabilize the T state break and some new ones form https://www.youtube.com/watch?v=H3DHvJ_MEtk Changes in Conformation Near Heme  Hemoglobin Binds Oxygen Cooperatively hemoglobin myoglobin hemoglobin has a hybrid sigmoid binding curve for oxygen  Sickle Cell Anemia Is a Molecular Disease of Hemoglobin sickle cell anemia: – homozygous condition – single amino acid substitution (Glu6 to Val6) chains produces a hydrophobic patch Normal and Sickle Cell Hemoglobin deoxygenated hemoglobin becomes insoluble and forms polymers that aggregate normal hemoglobin remains soluble upon deoxygenation