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Amino Acids, Peptides and Proteins Dr AN Boa Section 1 - Introduction 1. Use alanine, CH3CH(NH2)CO2H, as an example amino acid to explain what is meant by the following terms: (i) (ii) (iii) amphoteric; zwitterion; isoelectric point. Comment on alanine’s solubility in water and in toluene. 2. Draw the structure for the following amino acid / peptide derivatives described in shorthand notation: (i) (ii) (iii) (iv) 3. Ac-Tyr-OEt Tyr(tBu)-OEt Glu(OtBu)OEt Pro.Lys(Ac).Gly Draw the structure of diaminopimelic acid when in solution at i) pH 1, and ii) pH 12. Sketch the acid-base titration curve for this amino acid and mark on this curve where you would expect to find the isoelectric point. HO2C CO2H NH2 4. NH2 Draw the structure of histidine when in solution at (i) pH 1, and (ii) pH 12. Sketch a fully labelled acid-base titration curve for this amino acid and mark on this curve where you would expect to find the isoelectric point. HO2C NH NH2 N Amino Acids, Peptides and Proteins Dr AN Boa Section 2 – Separation and purification of amino acids and protein 1. 2. 3. The following peptides 1-4 are subjected to electrophoretic analysis at pH 6.0. 1 Ala.Arg Ala is CH3CH(NH2)CO2H 2 Phe.Ala.Arg Arg is H2N(C=NH)NH(CH2)4CH(NH2)CO2H 3 Phe.Arg.Glu Glu is HO2C(CH2)2CH(NH2)CO2H 4 Phe.Glu.Glu Phe is PhCH2CH(NH2)CO2H (i) State the overall charge of each peptide at pH 6.0. (ii) Predict, with a brief explanation, which electrode they will move towards and the relative rate of migration of each peptide. The following peptides are subjected to electrophoretic analysis at pH 1.0, pH 6.0 and pH 12.0. At which pH will the separation be best and why? 1 Lys.Gly.Ala Ala is CH3CH(NH2)CO2H 2 Gly.Asp.Lys Asp is HO2CCH2CH(NH2)CO2H 3 Asp.Gly.Asp Gly is CH2(NH2)CO2H Lys is H2N(CH2)4CH(NH2)CO2H Describe briefly the technique of X, explaining why it is particularly useful for the separation of peptides and proteins. (i) X = SDS-PAGE (ii) X = anionic and cationic ion-exchange chromatography (iii) X = size exclusion (gel permeation) chromatography (iv) X = isoelectric focussing electrophoresis Amino Acids, Peptides and Proteins Dr AN Boa Section 3 – Chemical and enzymatic reactions of amino acids and peptide 1. What fragments are formed when the following peptide is treated with the enzymes/reagents (i)-(v) indicated. Asp.Arg.Val.Tyr.Met.His.Pro.Phe.Lys.Leu.Leu.Val.Tyr.Ser 2. (i) trypsin; (ii) chymotrypsin; (iii) cyanogen bromide; (iv) Edman’s reagent; (v) carboxypeptidase. What products are obtained if the tripeptide Gly.Lys.Ala is subjected to end group analysis using Sanger’s reagent? Alanine, Ala, is CH3CH(NH2)CO2H, Glycine, Gly, is CH2(NH2)CO2H, and Lysine, Lys, is H2NCH2CH2CH2CH2CH(NH2)CO2H. What is the major advantage of Edman’s approach to end group analysis over that of Sanger? 3. The peptide Asp.Gly.Ala.Asp is cleaved cleanly into two fragments when treated with 6M hydrochloric acid at room temperature over several hours. Deduce the structure of the two fragments and explain their formation. Ala is CH3CH(NH2)CO2H; Asp is HO2CCH2CH(NH2)CO2H and Gly is CH2(NH2)CO2H Amino Acids, Peptides and Proteins Dr AN Boa Section 4 – Polypeptide sequencing 1. The pentapeptide A has the amino acid composition: (Gly1, Ile2, Tyr2) Use this and the following information to establish the structure of A. A A A 2. (i) FDNB DNP-Ile (ii) total acid hydrolysis carboxypeptidase partial acid hydrolysis Tyr (the first amino acid released) Products include only three dipeptides Explaining fully your workings, determine the sequence of the polypeptide A from the following experimental results: (i) Total acid hydrolysis of A gives: A = (Ala, Arg, Gly, 2×Lys, Met, Phe, Pro, 2×Ser, Tyr, Val) (ii) Treatment of A with Edman’s reagent gives the thiohydantoin of Val. (iii) Treatment of A with carboxypeptidase first releases Ala. (iv) Treatment of A with cyanogen bromide gives two peptides B and C with composition: B = (Ala, 2×Lys, Phe, Pro, Ser, Tyr) C = (Arg, Gly, Met, Ser, Val) (v) Treatment of A with chymotrypsin gives three peptides D, E and F with composition: D = (2×Lys, Phe, Pro) E = (Arg, Gly, Met, Ser, Tyr, Val) F = (Ala, Ser) (vi) Treatment of A with trypsin gives three peptides G, H and I with composition: G = (Gly, Lys, Met, Tyr) H = (Ala, Lys, Phe, Pro, Ser) I = (Arg, Ser, Val) Amino Acids, Peptides and Proteins Dr AN Boa Section 5 - Peptide synthesis 1. Devise a synthesis of the peptide Lys.Ala.Asp (shown below) starting from its constituent suitably protected amino acids (mechanisms are not required). State key reagents needed for your synthesis. O H N H2N H2N N H 4 CO2H O CO2H 2. Draw the structure of peptide Ala.Pro.Asp.Gly and devise a synthesis for it starting from suitably protected amino acids. 3. Draw the structure of peptide Lys.Lys(ε→α)Asp and devise a synthesis for it starting from suitably protected amino acids. Note: (ε→α) here is to indicate that the side chain amine of the middle lysine is linked to the α-acid of the aspartic acid. 4. Why is racemisation an issue in peptide synthesis? What tactics/strategies can be used to minimise this problem? Section 6 – Protein structure 1. Place the following inter-strand bond/interactions in order of increasing strength: (i) Disulfide bond (ii) Hydrogen bond (iii) van der Waals (iv) Salt bridge Draw an example to illustrate each type of interaction. 2. Describe the difference between a parallel and antiparallel β-sheet. 3. Explaining what is meant by “3.613 helix” discuss the structure of the α-helix. 4. Explain what is meant when an α-helix is described as amphipathic and describe how such helices are important in determining the tertiary and quaternary structures in proteins.