Peter Lang Chapter 5 Assignment, Assignment is due on Sept. 25th Fill in the blank Proteins are polymers in structure and made of small subunits called alpha amino acids The central carbon or alpha carbon is sp3 hybridized and has tetrahedral structure. Except the amino acid glycine, all other amino acids have stereoisomers D form and L form, which are called enantiomers. Naturally occurring proteins have all their constituted amino acids in L stereoisomeric form. 1. Name the amino acids with nonpolar-aliphatic side chains. Show the 1 letter designation. Glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I) 2. Name the amino acids with nonpolar aromatic side chains. Show the 1 letter designation. Phenylalanine (F), Tyrosine (Y), and Tryptophan (W) 3. Name the amino acids with polar side chains. Show the 1 letter designation and pKa values of the side chains. Serine (S), Cysteine (C), Threonine (T), Asparagine (N), and Glutamine (Q) pKa values: Serine - n/a; Cysteine – 8.3; Threonine – n/a; Asparagine – n/a; Glutamine – n/a 4. Name the amino acids with basic side chains. Show the 1 letter designation and pKa values of the side chains. Histidine (H), Lysine (K), and Arginine (R) pKa values: Histidine – 6.0; Lysine – 10.0; Arginine – 12.5 5. Name the amino acids with acidic side chains. Show the 1 letter designation and pKa values of the side chains. Aspartic acid (Aspartate) (D) and Glutamic acid (Glutamate) (E) pKa values: Aspartic acid – 3.9; Glutamic acid – 4.2 6. Name the two rare genetically encoded amino acids. Selenocysteine and Pyrrolysine Peter Lang 7. Name three amino acids that undergo changes following posttranslational modification. Hydroxylation of proline Carboxylation of glutamate Acetylation of lysine 8. What is a peptide bond, how is it formed, and what are the specific characteristics of a peptide bond? A peptide bond links amino acids together. It’s covalently formed by the amide bond between a carboxylic and amino group. 9. Define oligopeptide, polypeptide, and protein. Oligopeptide - A molecule composed of amino acid residues linked by peptide bonds that is smaller than a protein, which is called a polypeptide; 3 – 15 residues. Polypeptide – consisting of a large number of amino acids bonded together in a chain, forming a part of the protein. (greater than 15 residues) Protein – large molecules consisting of one or more large chains of amino acids. (greater than 50 residues) 10. Explain the spatial arrangement of the amino acids in the peptide bond and indicate the exception. For the spatial arrangement of amino acids in a peptide bond, the trans-form is will be favored over the cis form because the R groups on adjacent alpha carbons will not sterically interfere. The exception here is the sequence X – Pro. X represents any other amino acid. For this bond, the cis-configuration is sometimes allowed 11. Name the following modified peptide and explain the effect of a change in pH on the following peptide composition. Peter Lang Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser At a low pH (pH = 0), all ionizable residues will be in their protonated forms. This means amino groups will be positively charged and carboxyl groups will have a zero charge; the overall charge will be +2. If the pH of the solution is raised by titration with a strong base, ionizable groups will lose protons at pH values that associated with their pKa values. As the pH of the solution increases, more groups become deprotonated and the net positive charge will decrease. As more base is added, the net charge will reach a net charge of -2 as the peptide becomes increasingly negatively charged and basic because both amino groups are neutral and both carboxyl groups are negatively charged. All in all, a small shift in pH can alter the charges on the surface, or in the active site, of a protein and will affect a protein’s stability and/or functions. 12. Explain the process for activation of a protein through proteolytic cleavage. 1. Proteolytic enzymes such as protease can be used to catalyze the hydrolysis of peptide bonds. These enzymes can cleave specific polypeptide bonds. For example, in order to synthesize insulin, proinsulin must be converted into insulin by means of proteolysis. First, Preproinsulin is synthesized, and then the leader sequence will be cleaved by protease which forms disulfide bonds. Lastly, the mature insulin molecule with A and B chain will be formed. 13. Solve the problem 4 (a) and (b) in your book. Peter Lang 4a. S – Y – S - M - E – H – F – R - W - G – K - P – V 4b. 1623.93 g/mol 14. A researcher is trying to purify a protein with a molecular weight of 125 KD and a positive surface net charge. The gene for this protein is cloned into E. coli using an expression vector. Design a purification scheme for this protein. To separate the desired protein from other cellular components, we use cell lysis to release the contents of the protein into a buffer solution. Then, we will centrifuge the solution to remove the insoluble cell material and extract the lysate (soluble proteins). The resulting solution from this will be combination of proteins and nucleic acids. To further purify the desired protein, we will use a combination of affinity chromatography (IMAC), anion exchange chromatography (IEC), and size exclusion chromatography (SEC). For IMAC, the solution will bind tightly until most contaminants are washed off, and the desired protein is eluted. IEC will then separate the proteins based on surface charge densities. Finally, SEC will be used to separate the proteins at 125 KD.