Protein Chemistry (Part 1) PDF

Protein CHEMISTRY (part 1) Ass.Prof.Dr. Amany Mohamed Salah El-Din Medical Biochemistry & Molecular Biology Faculty of Medicine-Helwan University  OBJECTIVES Amino acids Amino acids Structure and classification importance Amin...

Protein CHEMISTRY (part 1) Ass.Prof.Dr. Amany Mohamed Salah El-Din Medical Biochemistry & Molecular Biology Faculty of Medicine-Helwan University  OBJECTIVES Amino acids Amino acids Structure and classification importance Amino acids Properties  Amino Acids are the Building Blocks of Proteins AA AA AA AA AA AA One Protein Amino Acid AA AA AA AA AA The 20 Key Amino Acids More than 300 amino acids occur naturally, but 20 of them that make up proteins. These 20 amino acids are the building blocks of proteins. Amino acid structure Amino acids are fatty acids which have a substituted amino group Each amino acid has 4 different groups attached to α- carbon ( which is C-atom next to COOH). These 4 groups are :  Amino group (NH2)  Carboxyl group(COOH)  Hydrogen atom R  Side Chain (R) Amino acid structures differ at the side chain (R-groups). Amino acids are α- amino acids (The amino group attached to the α- carbon which is C-atom next to COOH group). L-amino acids (The amino group is on left side configuration) Some D-amino acids occur in nature, but not in proteins. α- L-Form Amino Acid Structure Carboxylic group - COO Amino group + H3N a H R Amino acids classification  Classification of Amino Acids Chemical Based on different methods According Polarity of the side chain to polarity Based on whether amino acid can Nutritional be synthesized or not in the body Based on the fate of amino acids Metabolic in the body Chemical Classification 1- According to number of carbon atoms. 2- Amino acids and Imino acid. 3- Acidic , Basic and Neutral amino acids. 4- Amide group-containing amino acids. 5-Sulfur and Hydroxy containing amino acids. 6- Aromatic ,Heterocyclic and Aliphatic AAs. 7- Branched and Nonbranched chain AAs. According to number of carbon atoms 2 Carbons containing A.A Glycine 3 Carbons containing A.A Alanine Serine Cysteine  Phenyl alanine Tyrosine Tryptophan  Histidine 4 Carbons containing A.A Methionine Threonine 5 Carbons containing A.A Arginine Valine 6 Carbons containing A.A Lysine Leucine Isoleucine Imino acid: Proline Acidic , Basic and Neutral amino acids: Acidic Amino Acids (di-carboxylic groups)  Basic Amino Acids (di-amino groups) Amide group-containing amino acids: e.g. Glutamine and Asparagine Sulfur containing amino acids: Cysteine Methionine Hydroxy amino acids: Serine Tyrosine Threonine Aromatic amino acids (contain benzene ring) Phenyl alanine Tyrosine Tryptophan Heterocyclic amino acids(contain other type of ring) Histidine Proline Tryotophan Aliphatic amino acids (contain No ring) Other amino acids Branched chain amino acids Valine Leucine Isoleucine N.B Derived Amino Acids: β-alanine in vitamin structure. γ-amino butyric acid (GABA) neurotransmitter. Hydroxy-proline, hydroxy-lysine found in proteins.(structure of collagen) Ornithine, Citrulline in urea cycle.  Cystine formation of 2 mlecules of cysteine. Classification based on Polarity of side chain Amino Acids Hydrophilic aa Hydrophobic aa Basic side chain aa Acidic side chain aa Polar, uncharged side chain aa Polar (Hydrophilic) side chain They have polar groups in their side chain that can participate in hydrogen bond formation with water. They include ; uncharged side-chain (OH-SH-Amide): e.g. Serine, Threonine, Tyrosine, Cysteine, Asparagine and Glutamine.  charged side-chain a) Amino acids with a positively (+ve) charged side-chain (basic amino acids) Lysine, Arginine and Histidine. b) Amino acids with a negatively (-ve) charged side-chain (acidic amino acids) Glutamic acid and Aspartic acid. Non-polar (Hydrophobic side-chain): Other amino acids with a side chain that does not participates in hydrogen bonds formation with water.  Classification based on nutritional requirements (Biological classification) i) Essential amino acids: These amino acids cannot be synthesized in the body and have to be present essentially in the diet. e.g. Valine, Isoleucine, Leucine, Lysine, Arginine, Methionine, Threonine, Tryptophan Phenylalanine, and Histidine. (VITAL LYMPH) ii) Semi-essential amino acids: These amino acids can be synthesized in the body but the rate of synthesis is lesser than the requirement (e.g. during growth, repair or pregnancy) e.g. Arginine and Histidine. iii) Non-essential amino acids: Other amino acids that are synthesized in the body, thus their absence in the diet does not affect the growth. e.g.- Other remaining amino acids. Essential AA Nonessential AA Isoleucine Alanine Leucine Asparagine Lysine Aspartic Acid Methionine Cysteine Phenylalanine Glutamic acid Threonine Glutamine Tryptophan Glycine Valine Proline Arginine Serine Histidine Tyrosine Selenocysteine – the 21st amino acid as already stated, 20 amino acids are commonly found in proteins. In recent years, a 21st amino acid namely selenocysteine has been added. It is found at the active sites of certain enzymes/proteins (selenoproteins) e.g. glutathione peroxidase, glycine reductase, 5′-deiodinase, thioredoxin reductase. Selenocysteine is an unusual amino acid containing the trace element selenium in place of the sulfur atom of cysteine.  Biological value of protein When a protein contains the essential amino acids, it has a high biological Value. When one or more of the essential amino acids are missing or present in low numbers, the protein is has a low biological value. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids.  Classification based on metabolic fate The carbon skeleton of amino acids can be used either for glucose production , ketone bodies production Or both  Pure ketogenic Leucine and Lysine  Glucogenic and ketogenic (Mixed) Isoleucine, Tyrosine, Phenylalanine and Tryptophan  Pure Glucogenic The remaining amino acids are glucogenic  Properties of Amino Acids Soluble in Amphoteric Colorless polar solvent Property Isoelectric Buffering point activity 1-Amino Acids Solubility Soluble in polar Insoluble in solvents like non polar water due to solvents presence of charged groups  2-Amino acids are colorless But colorless Amino Aromatic acids do amino acids not absorb absorb visible light ultraviolet light (wave length 280nm)  3-Amphoteric Property The property of amino acid to COO - a behave as an acid +H (proton donor) or 3N H as a base (proton acceptor) R group  Amphoteric property Alkaline Carboxyl group donates a proton Amino acid becomes negatively charged media(low H AA migrates to the anode in an electric concentration) field. Acidic Amino group accept a proton Amino acid becomes positively charged media(high H AA migrates to the cathode in an electric concentration) field. Acidic environment Neutral environment Alkaline environment NH2 H+ NH2 H+ NH2 R-C-H R-C-H R-C-H COOH COO- COO- +1 0 -1 Isoelectric point Juang RH (2004) BCbasics 4-Isoelectric point  4-Isoelectric point At certain pH, which is specific for each amino acid, it carries equal positive and negative charges. This pH is called “isoelectric point’ and the amino acid molecule is called “Zwitter ion” or “dipolar ion” or “hybrid ion”  (5) Formation of peptide linkage Peptide bond formation is the most important reaction of amino acids. - The peptide bond is formed by condensation of the carboxyl group of one amino acid with the amino group of another amino acid. - L- alpha AA linked by peptide bonds form peptides. Many have physiologic activity including: Glutathione is a tripeptide formed of 3 amino acids (glutamic acid, cysteine and glycine). Glutathione and the enzyme glutathione reductase participate in the formation of the disulfide bonds of many proteins and polypeptide hormones and participate in the metabolism of xenobiotic.  Summary 1- Biological classification of amino Acids: Essential and non-Essential 2- Metabolic classification of Amino Acids glucogenic, ketogenic, mixed 3- By polarity : polar (charged- non charged) and non polar 4-Properties of Amino Acids: -Amphoteric property In a Basic solution (Lower Hydrogen Concentration) AAs Carry Negative charge. Acidic solution (high hydrogen Concentration)AAs carry Positive charge. - Isoelectric point (I.E.P) The specific pH at which the AA can exist in dipolar form with zero net charge.  What term describes molecules such as FAs that have both hydrophobic and hydrophilic regions, as shown? What does it mean for a FA to be unsaturated? What is the structural meaning of the designation “ Ꞷ-6” for an unsaturated FA?  The term amphipathic describes molecules such as FAs that have both hydrophobic and hydrophilic regions.  An unsaturated FA has one (monounsaturated) or more (polyunsaturated) double bonds in the cis configuration that causes a kink or bend in the molecule. If polyunsaturated, the double bonds are spaced at three-carbon intervals. An Ꞷ-6 unsaturated FA has a double bond six carbons from the methyl ( Ꞷ) end, as shown for arachidonic acid [20:4(5,8,11,14)]. References Course Notes: Lectures & labs Essential Book: Lippincott’s Illustrated Biochemistry Reviews (2017), 7th edition Recommended Book: Harper’s Illustrated Biochemistry (2018), 31st edition Periodicals, Web Sites: www.medscape.com, www.ekb.edu.eg Protein CHEMISTRY (part 2) Prepared by: Ass. Prof. Amany Mohamed Salah El-Din Wahb Assistant professor of Medical Biochemistry and Molecular Biology, Medical Biochemistry and Molecular Biology Department, Faculty of medicine, Helwan University Definition of Proteins: The proteins can be defined as the nitrogenous macromolecules composed of many amino acids. Biomedical or Clinical Importance: 1. Proteins are main structural component of cytoskeleton. 2. All enzymes which are called biological catalysts are proteinous in nature. 3. Proteins called as immunoglobulins serve as first line of defence against bacterial and viral infection. 4. Several hormone are proteinous in nature. These regulate many aspects of cell functions 5. Structural protein provide mechanical support and some proteins are called as contractile proteins, e.g. actin and myosine provide movements to muscles and therefore to body. Biomedical or Clinical Importance: 6. Some proteins are present in cell membrane, cytoplasm and nucleus which are called as receptors. They bind specific substances such as vitamins, harmones, etc. and mediate their cellular action. 7. The transport protein carry out the function of transporting specific substances either across the membrane or body fluids. 8. Storage proteins bind with specific substances and store them, e.g. iron is stored as ferritin. 9. Few Proteins are constituents of respiratory pigments and occur in electron transport chain or respiratory chain, e.g. cytochromes, Hb, myoglobins 10. Under certain conditions proteins can be catabolized to supply energy when lipids carbohydrates stores of body are exhausted. 11. Proteins by means of exerting osmotic pressure help in maintenance of electrolyte and water balance in body. CLASSIFICATION OF PROTEIN  Classification of Proteins Based on Chemical Nature 1. Simple protein: They are composed of only amino acid residue. 2. Conjugated proteins: Besides amino acids these proteins contain a non-proteinous moiety called as prosthetic group or conjugating group. 3. Derived proteins: These are denatured or degraded products of simple or conjugated proteins.  Protein Structure Primary Structure Secondary Structure Tertiary Structure Quaternary Structure  Protein Structure Primary Assembly STRUCTURE PROCESS Secondary Folding Tertiary Packing Quaternary Interaction  peptides: Peptides are compounds formed of less than 50 amino acids linked together by peptide bond. Proteins: The term protein is applied to describe molecules greater than 50 amino acids united together by peptide bonds. Some proteins are formed of 2 or more polypeptide chains. A specific sequence of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a protein is unique to that protein, and defines the structure and function of the protein.  Primary Structure Arrangement of the 20 amino acids in the polypeptide is the amino acid sequence which composes the primary structure of the protein The primary structure is held together by covalent or peptide bonds. The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) Counting of residues always starts at the N-terminal end(NH2-group). The primary structure of a protein National Genome Research Institute is determined by the gene genome.gov corresponding to the protein.  Primary Structure linear ordered 1 dimensional sequence of amino acid polymer By convention, written from amino end to carboxyl end A perfectly linear amino acid polymer is neither functional nor energetically favorable  folding!  Peptide Bonds It is a covalent bond formed between the carboxyl group of one amino acid and alpha amino group of another. It is formed by removal of water. Peptide formation needs energy getting from hydrolysis of high energy phosphate compound. No free rotation can occur around bond axis. http://web.mit.edu/esgbio/www/lm/proteins/peptidebond.html Peptide Bonds  Determination of amino acid sequences Each type of protein has a unique amino acid sequence. Different teqniques used for sequencing: a)Edman reaction b)molecular biology c)Mass spectrometry A)Edman reaction THE EDMAN REACTION ENABLES PEPTIDES & PROTEINS TO BE SEQUENCED phenylisothiocyanate(Edman’s reagent) selectively label the amino-terminal residue of a peptide. The resulting phenylthiohydantoin (PTH) derivative can be removed under mild conditions to generate a new amino terminal residue Successive rounds of derivatization with Edman’s reagent can therefore be used to sequence many residues of a single sample of peptide. B) Molecular biology: Knowledge of DNA sequences permits determination of the primary structures of polypeptides. DNA sequencing requires only minute amounts of DNA and can readily yield the sequence of hundreds of nucleotides DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of proline and lysine, and disulfide bond formation that accompany maturation.  Secondary Structure Secondary structure is the spatial relationship of adjacent amino acid residues (first and fourth ). These secondary structures are defined by patterns of hydrogen bonds between the main- chain peptide groups. Two main types of secondary structure the alpha helix and the beta sheet.  Secondary structure α-helix β-sheet Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.  Alpha Helix In this structure the polypeptide backbone is tightly turn around an imaginary axis and the R groups of the amino acid residues protrude outward from the helical backbone. Each helix turn includes 3.6 amino acid residues. The helical twist of the a` helix found in all protein is right handed. The structure is stabilized by a hydrogen bond between the hydrogen atom attached to the electropositive amide nitrogen and the electronegative carbonyl oxygen of the fourth amino acid on the amino terminal side. Not all polypeptides can form a stable a` helix.  Alpha Helix http://cmgm.stanford.edu/biochem/biochem201/Slides/  Hydrogen bond A hydrogen bond is a type of attractive force that exists between two opposite electric charged molecules In proteins, hydrogen bonds form between the backbone oxygen and amide hydrogen Although stronger than most other intermolecular forces, the hydrogen bond is much weaker than both the ionic bond and the covalent bond. When the spacing of the amino acid residues participating in a hydrogen bond occurs regularly between positions 1 and 4(1-4)an alpha helix is formed. When two strands are joined by hydrogen bonds involving alternating residues on each participating strand, a beta sheet is formed. Hydrogen bond  Beta Sheet http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/sheet.gif  Beta Sheet Features It is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment). They can be arranged side by side to form a structure resembling a series of pleats Beta Structures http://broccoli.mfn.ki.se The random coil: - Regions of proteins that are not organized as helices or pleated sheets are said to be present in random coil conformation. - Regions of random coil are of equal biologic importance to those of a- helix or - pleated sheet. Supersecondary structures (motifs): The intermediate in scale between secondary and tertiary structures are often termed supersecondary structures or motifs. It is typically formed of two secondary structures and a turn or loop and can be associated with particular function like Helix-loop-helix motifs provide the oligonucleotide-binding portion of many DNA-binding proteins such as repressors and transcription factors.  Tertiary Protein Structure Defines the three dimensional conformation of an entire peptide chain in space. Determined by the primary structure. Nearly all of the polar, hydrophilic R groups are located in the surface, where they may interact with water The nonpolar, hydropobic R groups are usually located inside the molecule  Types of Tertiary Structure Globular Disordered Fibrous Strong secondary Many insoluble Interacts well with structure allows amino acids, protein water and takes up a protein to retain a tends to minimize random configuration non-spherical shape surface/volume ratio Example: a keratin and collagen  Domains Domain – Portion of a protein that has a tertiary structure of its own. In larger proteins each domain is connected to other domains by short flexible regions of polypeptide.  Aspects which determine tertiary structure DISULFIDE BOND:  Covalent disulfide bonds formed between closely aligned cysteine residues form the unique Amino Acid cystine.  The process of chemical oxidation that forms interchain disulfide bonds can produce stable, covalently linked protein dimers, multimers or complexes, whereas intrachain disulfide bonds can contribute to C SH HS C protein folding and stability. H2 H2 [O] C S S C H2 H2 Hydrophobic interaction: CH3-CH3-  hydrophobic interaction refers to the tendency of non polar compounds to self-associate in an aqueous environment. They tend to be located in the interior of the polypeptide molecule.  Self-association arises from the need to minimize energetically unfavorable interaction between non polar groups and water.  The hydrogen bonds: NH-O=C Formed between hydrogen and oxygen atoms of the peptide bonds themselves and those formed between polar residues on the surface of proteins and water. The hydrogen bonds play important roles in the maintenance of protein structure above the primary order.  Electrostatic (ionic) bonds: -NH3+-O-OC These are salt bonds formed between oppositely charged groups in the side chains of amino acids e.g. interaction between the epsilon amino group of lysine (bears net charge of+1) and non alpha- carboxyl group of aspartate (bears a net charge of -1).  Quaternary Structure Quaternary structure is a larger assembly of several polypeptide chains, usually called subunits. The quaternary structure is stabilized by the same non- covalent interactions including hydrogen, hydrophobic and electrostatic bonds as the tertiary structure Or interchain covalent bonds like disulfide bonds.  Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin. Quaternary Structure non-linear 3 dimensional  Characteristics of denaturation 1. The native helical structure of protein is lost. 2. The primary structure of a protein with peptide linkages remains intact i.e., peptide bonds are not hydrolysed. 3. The protein loses its biological activity. 4. Denatured protein becomes insoluble in the solvent in which it was originally soluble.  Characteristics of denaturation 5. Denatured protein is more easily digested. This is due to increased exposure of peptidebonds to enzymes. Cooking causes protein denaturation and, therefore, cooked food (protein) is more easily digested. Further, denaturation of dietary protein by gastric HCl enchances protein digestion by pepsin.  Characteristics of denaturation 6. Denaturation is usually irreversible. For instance, omelet can be prepared from an egg (protein-albumin) but the reversal is not possible. 7. Careful denaturation is sometimes reversible (known as renaturation). Hemoglobin undergoes denaturation in the presence of salicylate. By removal of salicylate, hemoglobin is renatured. References Course Notes: Lectures & labs Essential Book: Lippincott’s Illustrated Biochemistry Reviews (2017), 7th edition Recommended Book: Harper’s Illustrated Biochemistry (2018), 31st edition Periodicals, Web Sites: www.medscape.com, www.ekb.edu.eg