Proteins PDF - MSU-Iligan Institute of Technology

Proteins Kirstin Rhys S. Pueblos, M.Sc. 1st sem AY 2024-2025 Department of Chemistry College of Science and Mathematics MSU-Iligan Institute of Technology  Outline: Amino Acids Classification of Amino Acids Essential Amino Acids Complementary Proteins Isoelectric...

Proteins Kirstin Rhys S. Pueblos, M.Sc. 1st sem AY 2024-2025 Department of Chemistry College of Science and Mathematics MSU-Iligan Institute of Technology  Outline: Amino Acids Classification of Amino Acids Essential Amino Acids Complementary Proteins Isoelectric Points of some amino acids Formation of Peptides Naming of Peptides Biologically Important Peptide BIOCHEMISTRY | Page 2  Outline (Continued) Polypeptides in the body Denaturation of Proteins: Temperature, pH, Organic solvents, Detergents, Heavy metals, Agitation Classification of Proteins Levels of Protein Structures: Primary, Secondary, Tertiary, Quaternary BIOCHEMISTRY | Page 3  Name protein is derived from the Greek word proteios which means “of first importance.” Proteins are polymers of amino acids Provide structure in membranes Build cartilage and connective tissues Transport oxygen in blood and muscle Direct biological reactions as enzymes Defend the body against infection Control metabolic processes as hormones BIOCHEMISTRY | Page 4  General structure of an α -amino acid BIOCHEMISTRY I | Page 5  Stereoisomers of Amino Acids a-carbon of amino acids is chiral glycine is the only common amino acid that is not chiral amino acid configuration isolated from proteins is L- the most oxidized end of the molecule, carbonyl, is drawn at the top BIOCHEMISTRY I | Page 5  The 20 Amino Acids in Proteins BIOCHEMISTRY | Page 7  Amino Acids BIOCHEMISTRY I | Page 8  Amino Acids BIOCHEMISTRY 1 | Page 9  Amino Acids BIOCHEMISTRY | Page 10  Amino Acids Derived amino acids (nonstandard amino acids) ➔ formed by enzyme-facilitated reaction o 4-hydroxyproline and 5-hydroxylysine BIOCHEMISTRY | Page 9\11  Amino Acids γ-carboxyglutamic acid O-phosphoserine - - constituent of several presence of P proteins involved in regulates the activity blood clotting. of proteins. BIOCHEMISTRY | Page 12  Amino Acids o cystine - made up of two cysteine molecules or residues joined together by a disulfide bond. The disulfide bonds play a special role in the structures of many proteins (e.g. hair) by forming covalent cross-links. o ornithine and citrulline - metabolites of the urea cycle BIOCHEMISTRY | Page 13  Essential Amino Acids BIOCHEMISTRY | Page 14  Examples of Complete and Incomplete Protein Sources BIOCHEMISTRY | Page 15  All the essential amino acids are provided in a meal that consists of a complete protein of animal origin or complementary proteins of vegetable origin. BIOCHEMISTRY I | Page 16  Properties of Amino Acids Nonionic and Zwitterionic forms of amino acids Figure 5. Nonionic and zwitterionic forms of amino acids. The nonionic form does not occur in significant amounts in aqueous solutions. The zwitterion predominates at neutral pH. BIOCHEMISTRY | Page 17.. Isoelectric Points of some amino acids BIOCHEMISTRY | Page 18  @ pH lower than IpH ➔ the solubility of aa increases (carboxylate end picks H+) ➔ the aa acquires net + charge; ➔ it migrates to cathode (-) electrode BIOCHEMISTRY | Page 19  @ pH higher than IpH ➔ the solubility of aa increases (H+ is removed from ammonium) ➔ the aa acquires net - charge; ➔ it migrates to anode (+) electrode BIOCHEMISTRY | Page 20  Summary of the changes in ionic charges when acid or base is added to a solution of amino acid at isoelectric point Isoelectric pH (IpH) ➔ the pH at which the + and – charges are balanced. BIOCHEMISTRY | Page 21  pKa Values for the Ionizing Groups of the Amino acid BIOCHEMISTRY | Page 22  Titration of ala +1 0 -1 pI= 2.3 + 9.7 2 pI = 6.0 BIOCHEMISTRY | Page 23  Titration of Glu +1 0 -1 -2 pI= 2.2 + 4.3 2 pI = 3.25 BIOCHEMISTRY | Page 24 BIOCHEMISTRY I | Page 25  BIOCHEMISTRY BIOCHEMISTRY | Page 26 BIOCHEMISTRY | Page 27 BIOCHEMISTRY | Page 28 BIOCHEMISTRY | Page 29 BIOCHEMISTRY | Page 30 BIOCHEMISTRY | Page 31  (+2)-------(+1)----------0--------- (-1)--------- (-2) 2.34 3.86 8.95 10.79 BIOCHEMISTRY | Page 32  Dipeptides Condensing or dehydrating two amino acids produces a dipeptide amino acid with a free a-NH3+ group is the amino terminal amino acid, N-terminal for short amino acid with a free -COO- group is the carboxyl terminal amino acid, C-terminal for short structures are written with the N-terminal on the left BIOCHEMISTRY | Page 33  Peptide Bond Proteins are linear polymers of L-α-amino acids carboxyl group of one amino acid is linked to the amino group of another amino acid linkage is an amide bond or peptide linkage this reaction is a dehydration reaction as water is released BIOCHEMISTRY | Page 34 Formation of a dipeptide | Page 35  Formation of a tripeptide BIOCHEMISTRY | Page 36  The Peptide Bond is Rigid and Planar The carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric dipole. Virtually all peptide bonds in proteins occur in this trans configuration BIOCHEMISTRY | Page 37 BIOCHEMISTRY | Page 38 BIOCHEMISTRY | Page 39  Some Biologically Important Peptides BIOCHEMISTRY | Page 40  Levels of Protein Structure BIOCHEMISTRY | Page 41 BIOCHEMISTRY | Page 42  The Primary Structure of Proteins primary structure is the amino acid sequence of the polypeptide chain - a result of covalent bonding between the amino acids – the peptide bonds each protein has a different primary structure with different amino acids in different places along the chain the 1o structure of proteins are translations of information contained in genes BIOCHEMISTRY | Page 43  Example of primary structure of protein: BIOCHEMISTRY | Page 44 BIOCHEMISTRY | Page 45  Normal 𝛽 𝑐ℎ𝑎𝑖𝑛: Val-His-Leu-Thr-Pro-Glu-Glu-Lys- Sickled 𝛽 𝑐ℎ𝑎𝑖𝑛 : Val-His-Leu-Thr-Pro-Val-Glu-Lys- BIOCHEMISTRY | Page 46  Amino Acid Sequencing HYDROLYSIS A. Acid Hydrolysis involves heating in the presence of 6N HCl in sealed tube at 110oC for 10 – 100 hrs. depending on the nature of peptide or protein to be hydrolyzed the protein is completely hydrolyzed, but trp, is destroyed completely and ser, thr, and tyr are partially destroyed. B. Alkaline Hydrolysis heating in the presence of 4N NaOH in sealed tube at 10 – 100 hrs as in acid hydrolysis does not damage trp, but destroys arg, cys, cys-cys, thr, & ser; and some amino acids are partly deaminated more disadvantageous but since it does not destroy trp, it is used in quantitative determination of this amino acid BIOCHEMISTRY | Page 47  HYDROLYSIS C. Enzymatic Hydrolysis (by proteases/ peptidases) Exopeptidases - cleaves external peptide bonds Aminopeptidases - sequentially cleaves peptide bonds, beginning at the N- terminal end of the polypeptide. - the liberated amino acids are identified one by one. BIOCHEMISTRY | Page 48  Amino Acid Sequencing Steps: 1. Cleavage of all disulfide bonds–oxidation with performic acid is commonly used. 2. Determination of the N–terminal amino acid. a. Sanger’s method - the polypeptide chain is reacted with 1–fluoro–2,4–dinitrobenzene (DNFB). The resultant dinitrophenyl-amino acid or DNP-amino acid can be separated from the other amino acids by ion – exchange chromatography after the polypeptide is hydrolyzed because it is more soluble in nonpolar solvents BIOCHEMISTRY | Page 49 BIOCHEMISTRY | Page 50 b. Edman degradation - uses phenylisothiocyanate (PITC), (Edman’s reagent.) which combines with the N-terminal amino acid to yield a phenylthiohydantoin-compound (PTH- compound). This can be identified by chromatography and extracted by organic solvent. | Page 51  3.Determination of the C–terminal amino acid A group of enzymes called the Carboxypeptidases are used to identify the C – terminal residue. Caboxypeptidases A and B, both secreted by the pancreas, hydrolyze peptides one residue at a time from the C–terminal end. a. Carboxypeptidase A – preferentially cleaves peptide bonds when an aromatic amino acid is the C–terminal residue. b. Carboxypeptidase B – cleaves basic amino acid residues. Because these enzymes sequentially cleave peptide bonds starting at the C–terminal residue, the first amino acid liberated is the C–terminal residue. BIOCHEMISTRY | Page 52  C–terminal amino acid c. Hydrazine method - hydrazine reacts with all amino acids whose carboxyl group is bound in peptide linkage, creating amino acylhydrazides. Only the C-terminal amino acid is spared. BIOCHEMISTRY | Page 52  4. Cleavage of the polypeptide into fragments a. Trypsin -cleaves peptide bonds on the carboxyl side of the two strongly basic amino acids arg and lys. b. Chymotrypsin-cleaves peptide bonds on the carboxyl side of the three aromatic amino acids (phe, tyr, and trp). c. Elastase - cleaves on the carboxyl side of gly and ala d. Pepsin - cleaves peptide bonds at the amino end of aromatic amino acids (phe, trp, tyr), acidic amino acids (asp, glu) and ile e. Thermolysin- cleaves peptide bonds at the amino end of the three aromatic amino acids, phe, tyr, trp; and amino acids with bulky nonpolar R groups, leu, ileu, and val.. BIOCHEMISTRY | Page 53  Cyanogen Bromide (CNBr) - specifically cleaves peptide bonds on the carboxyl side of methionine residues BIOCHEMISTRY | Page 55  Summary: BIOCHEMISTRY | Page 56  5. Ordering the peptide fragments The amino acid sequence information derived from two or more sets of polypeptide fragments are next examined for overlapping segments. Such segments make it possible to piece together the overall sequence. Significance of sequence analysis 1. Essential for understanding the protein’s mechanism of action (biological activity) 2. Important for the study of gene structure 3. Recognition of amino acid sequence has led to the chemical synthesis of medically useful polypeptide. BIOCHEMISTRY | Page 57  Sample Problems: 1. Consider the following peptide: Gly – Ile – Glu – Trp – Thr – Pro – Tyr – Gln – Phe – Arg – Lys What amino acids and peptides are produced when the above peptide is treated with each of the following reagents? a. Carboxypeptidase b. Chymotrypsin c. Trypsin d. DNFB BIOCHEMISTRY | Page 58  Solution: a. Because carboxypeptidase cleaves at the carboxyl end of peptides, the products are Gly – Ile – Glu – Trp – Thr – Pro – Tyr – Gln – Phe – Arg and Lys b. Because chymotrypsin cleaves peptide bonds in which aromatic amino acids (i.e., Phe, Tyr, and Trp) contribute a carboxyl group, the products are Gly – Ile – Glu – Trp, Thr – Pro – Tyr, Gln – Phe and Arg – Lys BIOCHEMISTRY | Page 59  c. Trypsin cleaves at the carboxyl end of lysine and arginine. The products are Gly – Ile – Glu – Tyr – Thr – Pro – Tyr – Gln – Phe – Arg and Lys d. DNFB tags the amino terminal amino acid. The products: DNP – Gly and Ile – Glu – Trp – Thr – Pro -Tyr – Gln – Phe – Arg – Lys BIOCHEMISTRY | Page 60  2. From the following analytical results, deduce the structure of a peptide isolated from the Alantian orchid that contains 14 amino acids. Complete hydrolysis produces the following amino acids: Gly (3), Leu (3), Glu (2), Pro, Met, Lys (2), Thr, Phe. Treatment with carboxypeptidase releases glycine. Treatment with DNFB releases DNP – glycine. Treatment with nonspecific proteolytic enzyme produces the ff: fragments: Gly – Leu – Glu, Gly – Pro – Met – Lys, Lys – Glu, Thr – Phe – Leu – Leu – Gly, Lys – Glu – Thr – Phe – Leu, Leu – Leu – Gly, Glu – Thr – Phe, Glu – Gly – Pro, Pro – Met – Lys – Lys, and Gly – Leu BIOCHEMISTRY | Page 61 Solution: The amino acid analysis provides information concerning the kind and number of amino acids in the peptide. The carboxypeptidase and DNFB results show that the carboxy and amino terminal amino acids are both glycine. Finally, by overlapping the fragments, the sequence of amino acids can be determined. Remember to start with a fragment that ends with the N – terminal residue, in this case, glycine. Gly – Leu – Glu, Gly – Pro – Met – Lys, Lys – Glu, Thr – Phe – Leu – Leu – Gly, Gly – Leu, Glu – Gly – Pro, Pro – Met – Lys – Lys, Lys – Glu – Thr – Phe – Leu, Leu – Leu – Gly The overall structure then becomes: Gly – Leu – Glu – Gly – Pro – Met – Lys ––Lys – Glu - Thr – Phe – Leu – Leu – Gly | Page 62 Exercises: 1. Hydrolysis of β–endorphin (a peptide containing 31 amino acid residues) produces the following amino acids: Tyr (1), Gly (3), Phe (2), Met, Thr (3), Ser (2), Lys(5), Gln (2), Pro, Leu (2), Val (2), Asn (2), Ala (2), Ile, His, and Glu Treatment with carboxypeptidase liberates Gln. Treatment with DNFB liberates DNP – Tyr. Treatment with trypsin produces the following peptides: Lys, Gly–Gln, Asn – Ala – Ile – Val – Lys, Tyr – Gly – Gly – Phe – Met – Thr – Ser – Glu – Lys, Asn – Ala – His – Lys, Ser – Gln – Thr – Pro – Leu – Val – Thr – Leu – Phe – Lys | Page 63 Treatment with chymotrypsin produces the following peptides: Lys – Asn – Ala – Ile – Val – Lys – Asn – Ala – His – Lys – Lys – Gly – Gln Tyr – Gly – Gly – Phe Met – Thr – Ser – Glu – Lys – Ser – Gln – Thr – Pro – Leu – Val – Thr – Leu – Phe What is the primary sequence of β–endorphin? 2. The following is the amino acid sequence of bradykinin, a peptide released by certain organisms in response to wasp stings: Arg – Pro – Pro – Gly – Phe – Ser – Pro – Phe – Arg What amino acids or peptides are produced when bradykinin is treated with each of the following reagents? a. Carboxypeptidase b. Chymotrypsin c. Trypsin d. DNFB | Page 64  The Secondary Structure of Proteins -Helix most common type of secondary structure coiled, helical important features: – each amide H and carbonyl O is involved in H bonds locking the helix in place – carbonyl O links to amide H 4 amino acids away – H bonds are parallel to the long axis of the helix – helix is right-handed – repeat distance or pitch is 5.4 angstroms – 3.6 amino acids per turn BIOCHEMISTRY | Page 65  -Helix BIOCHEMISTRY | Page 66  The Secondary Structure of Proteins BIOCHEMISTRY | Page 67 BIOCHEMISTRY | Page 68  -Helices in Fibrils fibrous proteins are arranged in a secondary structure of fibers or sheets with only 1 type of secondary structure repeated coiling of helices BIOCHEMISTRY | Page 69  -Helices in Fibrils Molecular structure of myosin. BIOCHEMISTRY | Page 70 | Page 71  β -pleated sheet formed when two or more polypeptide chain segments line up side by side. peptide backbone is in an extended conformation (β-strand ) 1. parallel β-pleated sheet N-termini are head to head. 2. antiparallel β -pleated sheet N-terminus of one chain is aligned with the C-terminus of a second chain (head to tail). BIOCHEMISTRY | Page 72  β -pleated sheet BIOCHEMISTRY | Page 73  β -pleated sheet BIOCHEMISTRY | Page 74  β -pleated sheet BIOCHEMISTRY | Page 75  Supersecondary structures BIOCHEMISTRY | Page 76  β-Turns - allows the peptide chain to reverse direction - also called β-bend, tight turn or reverse turn BIOCHEMISTRY | Page 77  β-Turns - carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away - proline and glycine are prevalent in beta turns (why?) BIOCHEMISTRY | Page 78 BIOCHEMISTRY | Page 79  not all amino acids are equally likely to be found in a given type of secondary structure BIOCHEMISTRY | Page 80 BIOCHEMISTRY | Page 81  The Tertiary Structure of Proteins The following types of interaction stabilize tertiary structure: Van der Waals forces between the R groups of nonpolar amino acids that are hydrophobic Hydrogen bonds between the polar R groups of the polar amino acids Ionic bonds (salt bridges) between the R groups of oppositely charged amino acids Covalent bonds between the thiol-containing amino acids. Two of the polar cysteines can be oxidized to a dimeric amino acid called cystine. The disulfide bond of cystine can be a cross-link between different proteins, or it can tie two segments within a protein together. BIOCHEMISTRY | Page 82  Types of Interactions Maintaining Tertiary Structure Disulfide bridges between two cysteine residues Salt bridges between ionic side chains -COO- and -NH3+ Hydrogen bonds between polar residue side chains Hydrophobic interactions: two nonpolar groups are attracted by a mutual repulsion of water BIOCHEMISTRY | Page 83  Summary of the weak interactions that help maintain the tertiary structure of a protein. BIOCHEMISTRY | Page 84 BIOCHEMISTRY | Page 85  Question: What type of interaction would you expect between the side chains of the following amino acids? 1. cys + cys 2. glu + lys 3. ala + val 4. pro + met BIOCHEMISTRY | Page 86  The Quaternary Structure of Proteins –association of several polypeptides to produce a functional protein. Figure. Structure of hemoglobin. The protein contains four subunits, designated α and β. The α- and β-subunits face each other across a central cavity. Each subunit in the tetramer contains a heme group that binds oxygen. BIOCHEMISTRY | Page 87  Exercises: Identify each of the following observations about protein structure as being aspects of the 1o, 2o, 3o, or 4o structure 1. Amino acids 14–38 in the sequence are arranged in α–helix pattern 2. When the protein was enzymatically broken apart, one of the fragments was found to be Leu-Tyr-Gly-Ala-Lys. 3. The entire molecule is globular 1.3 times as long in one dimension as its diameter. 4. Detergent was added to a water solution of the protein. The molecular weight study before the addition indicated a single protein with a molecular weight of 150,000. After the addition, two proteins were present with molecular weights 25,000 and 50,000. BIOCHEMISTRY | Page 88 | Page 89  Classification of Proteins Proteins can be loosely classified by shape & solubility Fibrous porteins - simple and regular linear structures - axial ration > 10 - serve mainly structural roles - low solubility in water or dilute salt solution Globular proteins - compactly folded, approx. spherical shape - axial ratio < 10(but usually not > 3-4) - serve mainly functional roles (e.g. enzymes) Membrane proteins - found in membranes; frequently polyhelical structures - insoluble in water; BIOCHEMISTRY | Page 90 BIOCHEMISTRY | Page 91  Classification of Proteins by Composition BIOCHEMISTRY | Page 92  Classification of Proteins by Function BIOCHEMISTRY | Page 93  Denaturation of Proteins Denaturation of a protein occurs when there is a change that disrupts the interactions between R groups that stabilize the secondary, tertiary, or quaternary structure. However, the covalent amide bonds of the primary structure are not affected. Temperature As the temperature continues to increase, the bonds within the proteins begin to vibrate more violently and eventually, the weak interactions, like hydrogen bonds and hydrophobic interactions, that maintain the protein structure are disrupted. BIOCHEMISTRY | Page 94  Figure 3. Denaturation of egg protein occurs when the bonds of the tertiary structure are disrupted. BIOCHEMISTRY | Page 95  Summary of the factors that denature protein and their effects. BIOCHEMISTRY | Page 96