1. CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Peptides - General structure and properties

2. CHMI 2227 - E.R. Gauthier, Ph.D. 2 Peptides Peptides are polymers of amino acids; Amino acids building blocks (residues) are linked to each other through a covalent bond: the peptide bond. 12 1 2 A dipeptide

3. CHMI 2227 - E.R. Gauthier, Ph.D. 3 Peptides Polypeptides and proteins are simply chains of amino acids linked together through peptides bonds:  If less than 20 residues: oligopeptide;  If more than 20 residues but molecular mass (Mr) < 10,000 Da: polypeptides.  If Mr > 10 kDa: protein. Particular terminology:  Dipeptide (2 residues) / Tripeptide (3 residues) / Tetrapeptide (4 residues) / Pentapeptide (5 residues) / Ect, ect, ect. Note: 1 Da (dalton) = 1 g /mol. Little trick: Mr of a polypeptide/protein ~ number of amino acids x 110 Da.

4. CHMI 2227 - E.R. Gauthier, Ph.D. 4 Peptides - polarity Each peptide has a polarity:  One extremity with the NH 2 (the one bonded to the C  ) which is not part of a peptide bond: N-terminal end;  One extremity with the COOH (the one bonded to the C  ) which is not part of a peptide bond: C-terminal end; By convention: the N-terminal is always placed on the left, and the C-terminal on the right.

5. CHMI 2227 - E.R. Gauthier, Ph.D. 5 Peptides - nomenclature Different ways to write this peptide (hyphen = peptide bond) :  Tyrosyl-glycyl-glycyl-phenylalanyl-leucine  Tyr-Gly-Gly-Phe-Leu  Y-G-G-F-L  YGGFL Note that the peptide is always written with the N-ter to the left and the C-ter to the right (NH 2  COOH).

6. CHMI 2227 - E.R. Gauthier, Ph.D. 6 Peptide: hydrolysis The composition (NOT the sequence) of a peptide in its amino acid constituents is determined by first hydrolysing the peptide bond, and then identifying the amino acids:  Tyr-Gly-Gly-Phe-Leu  Gly 2, Leu, Phe, Tyr The amino acids are then purified by High Pressure Liquid Chromatography (HPLC). Detection is done by UV absorbance. To detect those amino acids that cannot absorb UV (you know which ones…), the amino acid are derivatized, meaning they are chemically coupled with a compound that absorbs UV. Quantification and identification of the amino acids is often done with the help of standards (analyzed with the same system but in a separate experiment); 6 M HCl

7. CHMI 2227 - E.R. Gauthier, Ph.D. 7 Analysis of amino acids Detection of amino acids: ninhydrin reagent While Trp, Phe and Tyr can be detected by their A 260-280nm, the other amino acids cannot; Ninhydrin reacts with the amine group of amino acids, generating a purple product (yellow in the case of Pro). The ninhydrin reaction allows one to detect and quantify (A 570nm ) the amino acids contained in the fractions of the IEX column. O O OH Ninhydrin Amino acid 2 O O N O O C H R COO - NH 3 + CO 2 Purple!! R-HC=O

8. CHMI 2227 - E.R. Gauthier, Ph.D. 8 High Pressure Liquid Chromatography (HPLC) PITC = phenylisothiocyanate PTC = phenylthiocarbamyl http://www.protein.iastate.edu/aaa.html

9. CHMI 2227 - E.R. Gauthier, Ph.D. 9 High Pressure Liquid Chromatography (HPLC) The relative amount of each amino acid is given by calculating the area under each curve. http://www.protein.iastate.edu/aaa_figure3.html

10. CHMI 2227 - E.R. Gauthier, Ph.D. 10 Peptide - ionization Each peptide will exist in different protonated forms, depending on the pH and its amino acid composition:  Terminal amino and carboxyl groups can be protonated/ionized as in the free amino acid;  The side chain can also be ionized, if an appropriate group is present;  The NH 2 and COOH groups that are part of the peptide bond are NOT ionized. Therefore, there will be a pH where a given peptide/protein will carry no net charges: this pH value will be the isoelectric point of the peptide/protein in question. Example: Ionization of the peptide GAVFD at pH 2, 6 and 12.

11. CHMI 2227 - E.R. Gauthier, Ph.D. 11 Example of peptides 1. Aspartame: artificial sweetener 2. Oxytocin: stimulates uterine contractions Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Glycinamide residue: 2 HN-CH 2 -CONH 2 SS Disulfide bond H 3 N + -CH-C-NH-CH-C-OCH 3 COO - CH 2 O O Asp-Phe-methyl ester

12. CHMI 2227 - E.R. Gauthier, Ph.D. 12 Example of peptides 3.Insulin Intrachain disulfide bond Interchain disulfide bonds

13. CHMI 2227 - E.R. Gauthier, Ph.D. 13 Example of peptides 4.Cystic Fibrosis Transductance Regulator Single polypeptide chain of 1480 amino acids; Responsible for the transport of chloride ions across the cell membrane; Mutation of F508 yields a non-functional protein and cystic fibrosis. What is the approximate Mr of CFTR?

14. CHMI 2227 - E.R. Gauthier, Ph.D. 14 General properties of proteins 1. Proteins differ in their Mr. Protein Mr (kDa) # residues # chains Insulin5.7512 Cytochrome c131041 Ribonuclease A13.71241 Lysozyme13.91291 Myoglobin16.91531 Chymotrypsin21.62413 Chymotrypsinogen222451 Hemoglobin64.55744 Serum albumin68.55501 Hexokinase1028002 Immunoglobulin G1451,3204 RNA polymerase4504,1005 Apolipoprotein B5134,5361 Glutamate dehydrogenase 1,0008,30040 Source: Biochemistry. Lehninger.

15. CHMI 2227 - E.R. Gauthier, Ph.D. 15 Multimeric proteins Proteins with more than one polypeptide chains are called multimeric proteins; Different types of multimeric proteins exist:  Homo/oligomeric: 2 or more copies of the same polypeptide chain;  Heteromeric: different polypeptide chains make up the proteins. The different polypeptides of a multimeric protein (i.e. the protein’s subunits) can be held together in different ways:  Disulfide bonds  Hydrogen bonds  Hydrophobic interactions  Electrostatic interactions Multimeric proteins most often require all their parts in order to be functional. Very often proteins can change partners, providing them leading to their in/activation or giving them a different function. Monomer/subunit Homodimer 1 1 Heterodimer 1 2 Hydrogen bonds: N-H ----- O-H N-H ----- N O-H ----- O=C N-H ----- O=C Hydrophobic interactions: -CH 3 CH 3 - CH 3 Electrostatic interactions: COO - ----- H 3 + N

16. CHMI 2227 - E.R. Gauthier, Ph.D. 16 Importance of multimeric proteins – planar cell polarity

17. CHMI 2227 - E.R. Gauthier, Ph.D. 17 Importance of multimeric proteins – planar cell polarity Nature Genetics 38, 21 - 23 (2006)

18. CHMI 2227 - E.R. Gauthier, Ph.D. 18 General properties of proteins 2. Proteins differ in their pI. ProteinpI Pepsin1 Egg albumin4.6 Serum albumin4.9 Urease5  -lactoglobulin 5.2 Hemoglobin6.8 Myoglobin7 Chymotrypsinogen9.5 Cytochrome c10.7 Lysozyme11 Source: Biochemistry. Lehninger.

19. CHMI 2227 - E.R. Gauthier, Ph.D. 19 General properties of proteins 3. Frequency of amino acid composition Number of residues per molecule of protein Amino Acid Human cytochrome c Bovine chymotrypsinogen Ala622 Arg24 Asn515 Asp38 Cys210 Gln210 Glu85 Gly1323 His32 Ile810 Number of residues per molecule of protein Amino Acid Human cytochrome c Bovine chymotrypsinogen Leu619 Lys1814 Met32 Phe36 Pro49 Ser228 Thr723 Trp18 Tyr54 Val323 Total104245

20. CHMI 2227 - E.R. Gauthier, Ph.D. 20 General properties of proteins 3. Frequency of amino acid composition

21. CHMI 2227 - E.R. Gauthier, Ph.D. 21 General properties of proteins 4. Proteins can include other chemical groups in addition to amino acids ClassProsthetic group Example LipoproteinLipids  1-lipoprotein (blood) Glycoprotein Carbohydrates (sugars) Immunoglobulin G (blood) Phosphoprotein Phosphate groups Casein (milk) Hemoprotein Heme (iron porphyrin) Hemoglobin Flavoprotein Flavin nucleotides Succinate dehydrogenase Metalloprotein Fe Zn Ca Cu Ferritin Alcohol dehydrogenase Calmodulin Plastocyanin

22. CHMI 2227 - E.R. Gauthier, Ph.D. 22 General properties of proteins 5. Proteins have a specific shape Each polypeptide spontaneously adopts a shape or conformation. This conformation is unique to each protein; A protein in its correct conformation is said to be native; Disruptions in the conformation (e.g. by heating) denatures the protein and usually leads to its inactivation. Globular proteins Fibrillar (rod-like) proteins