Amino Acids, Proteins and Enzymes - PDF

AMINO ACIDS, PROTEINS AND ENZYMES  Key Concepts 1. Amino acids are the building blocks of proteins and they are made up primarily of carbon, hydrogen, oxygen, and nitrogen. 2. Each amino acid contains an amino group, a carboxyl group and an R-group (side chain). 3. Differences in...

AMINO ACIDS, PROTEINS AND ENZYMES  Key Concepts 1. Amino acids are the building blocks of proteins and they are made up primarily of carbon, hydrogen, oxygen, and nitrogen. 2. Each amino acid contains an amino group, a carboxyl group and an R-group (side chain). 3. Differences in R-group give rise to different amino acids and differences in properties of the amino acid. 4. Proteins can have up to 4 levels of structure (Primary, Secondary, Tertiary and Quaternary). 5. Primary structures (carbon backbones) are built from strong covalent bonds. 6. Secondary, tertiary and quaternary structures have mostly weak intermolecular interactions, hence protein structures are dynamic. 7. Protein function is determined by its structure. Changes in structure affect the properties and functions of the protein. Did you know? The egg white contains large quantities of protein. Under increased heat, the proteins lose their 3-D conformation and coagulate to from the whitish precipitate. Photo credits: Denise Neil retrieved from http://blogs.kansas.com/dining/2012/01/18/question-of-the-week-best-egg-creation/  Amino Group AMINO ACIDS Carboxyl Group Mainly made up of C, H, O, N contain R- Group * (side chain) make up primary Covalent bond PROTEIN - strong 4 levels of structure secondary Mostly weak tertiary intermolecular quaternary interactions can function as ENZYMES Dynamic structures leads to Different properties & functions Central Dogma of Molecular Biology  Introduction to amino acids Amino acids are the basic building blocks of proteins. Amino H Carboxyl group | group They contain a carboxyl group, an amino group and a side chain. H2N – C – COOH | The side chain (or R group) varies in R every amino acid. Side chain The side chain determines the property of different amino acids. Amino H Carboxyl group | group H3+N – C – COO- In aqueous solution, the amino group has the ability to accept a | H+ (proton), while the carboxyl R Side chain group has the ability to release a H+ (proton). Amino group can accept a proton from water and become positively charged. NH2 + H2O NH3+ + OH- Carboxyl group can release a proton into water and become negatively charged. COOH COO- + H+  The amino group is thus an organic base because bases can accept H+. H H | | H2N – C – COOH H3+N – C – COOH H+ | | R R The carboxyl group is thus an organic acid because an acid can donate H+. H H | | H2N – C – COOH H2N – C – COO- | | H+ R R Therefore, amino acids are amphoteric, because they contain BOTH acid and base functional groups. The Zwitterion and Isoelectric Point Under specific conditions, both the positive charge and the negative charge can exist in the same amino acid at the same time. We call this a zwitterion. H H H | | | H3+N – C – COOH H3+N – C – COO- H2N – C – COO- | | | R R R +1 Charge zwitterion -1 Charge Overall 0 Charge A zwitterion is an amino acid that has both positive and negative charges. A zwitterion has an overall net charge of zero. The charge an amino acid carries is determined by the pH of the solution the amino acid is in. For every amino acid, there exist a pH where both the positive and negative charges are present (i.e. the amino acid has overall zero charge). We call this the isoelectric point. The isoelectric point is the pH at which the zwitterion exists (i.e. the amino acid has overall zero charge). H H H | | | H3+N – C – COOH H3+N – C – COO- H2N – C – COO- | | | R R R +1 Charge zwitterion -1 Charge Overall 0 Charge Different amino acids usually have different isoelectric points.  What happens when pH is below the isoelectric point? (i.e. more acidic environment) In an acidic environment, H+ H+ there is a high concentration H of H+ protons. H+ | All the functional groups on H3+N – C – COOH the amino acid gets protonated. | R COO- + H+ → COOH H+ H+ NH2 + H+ → NH3+ H+ H+ Therefore, at pH below the isoelectric point, the amino acid becomes positively charged.  What happens when pH is above the isoelectric point? (i.e. more basic environment) In an alkaline (basic) environment, OH- there is a low concentration of H+ OH- H protons (or high in OH-). | All the functional groups on the H2N – C – COO- amino acid gets deprotonated. | COO- + H2O  COOH + OH- R H+ H+ NH2 + H2O  NH3+ + OH- OH- OH- OH- Therefore, at pH above the isoelectric point, the amino acid becomes negatively charged. DIFFERENT CLASSES OF AMINO ACIDS Allocating a Group number based on Polarity and Charge. The importance of the Thiol functional group (SH) on Cysteine. Classes of amino acids Group 1: Non-polar side chain Group 2: Polar, uncharged side chain Group 3: Polar, charged side chain There are 20 amino acids in nature. These can be classified based on their side chain (R-group). The alpha carbon H | H2N – C – COOH | R The alpha carbon (or Cα) is the carbon atom at the center. Or more correctly, it is the 1st carbon connected to the —COOH group  Find the Alpha carbon. Describe the R-group. R-group is: (i) non-polar, (ii) uncharged. File:Phenylalanin - Phenylalanine.svg R-group It contains an aromatic ring. C aromatic ring alpha carbon Is this R-group hydrophobic or hydrophilic? Classes of amino acids (example): Group 1: Non-polar; Uncharged R-group Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine Group 2: Polar, Uncharged R-group Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine Group 3: Polar, Charged R-group Aspartic acid, Glutamic acid, Lysine, Arginine, Histidine Do not need to memorise the examples Quick Summary: The R-group confers special characteristics to the amino acid. The R-group can be polar or non-polar charged or uncharged If charged, the charge can be positive or negative charge Self-check: Amino acids are the building blocks of a) carbohydrates b) nucleic acids c) lipids d) proteins Self-check: Amino acids has a) both amino group and carboxyl group b) both amino group and keto group c) amino group only d) carboxyl group only Self-check: Which statement about the zwitterionic form of an amino acid is correct? a) The zwitterion acts only as an acid b) The zwitterion acts only as a base c) The zwitterion carries an overall charge which can be positive or negative d) The zwitterion is neutral overall Self-check: The isoelectric point of an amino acids is defined as the pH a) where the molecule carries no electric charge b) where the carboxyl group is uncharged c) where the amino acid group is uncharged  Amphoteric Zwitterion Isoelectric point Amino Group AMINO ACIDS Carboxyl Group Make up of C, O, H, N contain R- Group * (side chain) make up Covalent bond primary - strong PROTEIN 4 levels of structure secondary Mostly weak tertiary intermolecular interactions quaternary can function as ENZYMES Dynamic structures leads to Different properties & functions  The peptide bond H H H O H | | | || | H3+N – C – COO- | + H3+N – C – COO- | → H3+N – C – C - N – C – COO- | | | CH3 CH3 CH3 H CH3 Amino acid 2 Amino acid 1 + H2O Is a covalent bond formed between an amino group from one amino acid and a carboxyl group from another amino acid. Formula: -C(=O)NH- During peptide bond formation, H20 is released (condensation reaction). The peptide bond General characteristics: δ- It is a covalent bond It consists of O, C, N and H atoms. δ- Properties: It is flat or planar. Has double bond characteristics - it is δ+ rigid. The bond cannot rotate freely around the C-N bond. It is polar. It can form hydrogen bonds with another peptide bond. δ+  Peptide, oligopeptide, polypeptide, protein Peptide bond _ N-terminus R1 R3 C-terminus - NH3+ Glu Ala Lys Ser COO R2 R4 Different amino acids may form peptide bonds and link up to form a sequence of amino acid residues. Peptide – a chain of amino acids joined by peptide bonds Oligopeptide – a short chain of amino acids (typically 2 - 20) Polypeptide – a long chain of amino acids (typically >20) Protein – one or more polypeptide chains that fold and form a 3-D structure Quick Summary: Peptide bond Covalent bond formed between carboxyl and amino group of two amino acids. Formula: -C(=O)NH- Joins amino acids into a peptide chain Disulfide bond Covalent bond formed between R-groups of two cysteine (thiol functional group). Formula: -S-S- Maintains structure of proteins Cysteine can form disulphide bonds Cysteine contains a thiol functional group on its side chain (R-group). H | Symbol for thiol is (-SH) H3+N – C – COO- | CH2 The -SH group is polar | SH Cysteine can form disulphide bonds The thiol on one cysteine and a H | thiol on another cysteine can H3+N – C – COO- react to form a disulphide bond. | CH2 Disulphide bonds are important in | maintaining the structure of S proteins. S | CH2 | H3+N – C – COO- | H NON-COVALENT INTERACTIONS Occurring between peptide bonds Occurring between R-groups Occurring between Terminal NH3+, COO- ends Non-covalent interactions Can attract (+) Can attract (-) Or repel (-) Or repel (+) Can attract (+) Or repel (-) Can _ form H-bond R R + + * * * - NH3 Glu Ala Lys Ser COO R R Can attract (-) Or Hydrophobic OH repel (+) interaction Can H-bond, and Can form H-bond interact with ionic charges * Peptide bonds can also form H-bond Non-covalent interactions Peptides interact with each other via non-covalent interactions occurring on: a) Peptide bonds (because of H-bonding) b) R-groups (H-bonding, hydrophobic interaction and ionic interaction) c) Terminal NH3+, COO- (H-bonding, ionic interaction) Examples a) The peptide bond can interact with other molecules via H-bonding. Amino acid 1 Amino acid 2 The partially negatively charged O atom on one δ+ peptide bond can H-bond H bond with the partially δ– positively charged H atom on another peptide bond. Amino acid 3 Amino acid 4 Examples b) R-groups can interact with other molecules via H-bonding, hydrophobic interactions and ionic interactions H | H3+N – C – COO- | CH2 | δ– OH δ+ E.g. Serine can E.g. The R-group on phenylalanine H-bond via the R-group can interact with other hydrophobic groups via hydrophobic interaction. Examples b) R-groups can interact with other molecules via H-bonding, hydrophobic interactions and ionic interactions H | The negatively charged O H atom on the R-group of H3+N – C – COO- glutamic acid can form ionic | | interaction with the NH3+ on CH2 H3+N – C – COO- the R-group of Lysine | | CH2 (CH2)4 | | C=O NH3+ | Lysine has a positively O- charged R-group Glutamic acid has a negatively charged R-group Examples c) Terminal NH3+ and COO- can interact with other molecules via H-bonding and ionic interactions H O H O H O | || | || | || H3+N – C – C - N – C – C – N – C – C – O – Peptide 1 | | | | | CH3 H CH3 H CH3 δ+ δ– H bond H O H O H O | || | || | || H3+N – C – C - N – C – C – N – C – C – O – | | | | | Peptide 2 CH3 H CH3 H CH3 Examples c) Terminal NH3+ and COO- can interact with other molecules via H-bonding and ionic interactions Peptide 1 H O H O H O | || | || | || Ionic interaction H3 N – C – C - N – C – C – N – C – C – O – + | | | | | CH3 H CH3 H CH3 H O H O H O | || | || | || H3+N – C – C - N – C – C – N – C – C – O – | | | | | Peptide 2 CH3 H CH3 H CH3 Intra- and inter-molecular interaction Amino acid residues on a peptide can interact non-covalently with other amino acid residues (i) within the same peptide (intra-molecular interaction) or (ii) across a different peptide (inter-molecular interaction) (i) Intra-molecular interaction Internal attraction of Intra-molecular interactions may side chains also occur along the entire sequence of the polypeptide. This allows the polypeptide to bend at different parts into a folded configuration. Ala Opposite charges on the side chains will attract, like charges will repel, and hydrophobic side chains Peptide 1 will group together. (ii) Inter-molecular interaction The peptides will align themselves to fit the Occurs between 2 or more peptides. maximum interactions possible, leading to a R R stable configuration. COO Ser Lys Ala Glu NH3+ - Peptide 1 R R + _ _ R R + + - Peptide 2 NH3 Glu Ala Lys Ser COO R R PROTEIN STRUCTURE Primary structure (1°) of proteins Secondary structure (2°) of proteins Tertiary structure (3°) of proteins Quaternary structure (4°) of proteins  Primary Structure The primary structure of a protein refers to the amino acid sequence of the polypeptide. By convention, the sequence is written with the N-terminus on the left and the C-terminus on the right. N-terminus C-terminus What is the primary structure of this peptide chain? Glu – Ala – Lys – Ser Primary Structure Secondary Structure Primary structure polypeptides can fold to form secondary structures: a. α-helix b. β-pleated sheets (or sometimes called beta-sheets) c. β -turns d. random coils These structures form because of hydrogen bonds. They give proteins strength and flexibility. Secondary Structure: Alpha Helix The α-helix is a polypeptide strand folded into a spring-like coil. Secondary Structure: Alpha Helix The α-helix is a polypeptide strand folded into a spring-like coil. Secondary Structure: α-helix Held by H-bonds that is formed between peptide bonds. The oxygen on the C=O has a partial negative charge. The hydrogen on the N-H has a partial positive charge. Every –NH in each peptide bond is H-bonded to the 4th peptide bond C=O group. Secondary Structure: Beta-pleated Sheet A β-pleated sheet is a secondary structure consisting of beta strands connected laterally by at least two or three H-bonds, forming a generally twisted, pleated sheet. Picture source: http://en.wikipedia.org/wiki/Beta_sheet Secondary Structure: β-pleated Sheet Every -C=O group on the peptide bonds of one polypeptide strand forms a H-bond with every –NH IIIIIII group on the peptide bond of the adjacent strand. IIIIIII β-strands are short polypeptides of 5-10 amino acids. IIIIIII IIIIIII β-strands are fully extended and usually twisted forming a sheet which is also twisted. Picture source: http://en.wikipedia.org/wiki/Beta_sheet Secondary Structure: β-pleated Sheet The beta strands are represented by arrows which point to the direction from N-terminal to C-terminal. Picture source: http://en.wikipedia.org/wiki/Image:BetaPleatedSheetProtein.png Secondary Structure: Parallel β-pleated Sheets Parallel β-sheet : The adjacent strands are aligned in the same direction. Secondary Structure: Anti-Parallel β-pleated Sheets Anti-parallel β-sheet : The adjacent β -strands are aligned in opposite directions. The -NH and –C=O groups on adjacent peptide bonds are thus aligned to each other. This results in a H-bonding pattern which is parallel and the lengths of the H-bonds are shorter. Thus the structure is more stable with stronger attraction compared to the strands in parallel beta-sheets.  Secondary Structure: β-turns and Random Coils 2nd 3rd PB PB H-bond may occur between peptide 1st 4th bonds that are PB PB between 3-5 amino acids apart. Hairpin turn is a β-turn where the Random coils are formed by H- polypeptide reverse direction and bonds of peptide bonds with is held by one or two H-bonds. functional groups on side chains, The H-bonds occur in the peptide or between side chains. bonds just like those in β-sheets. Tertiary Structure Proteins tend to fold spontaneously into a distinct tertiary structure. The tertiary structure is the final 3-D structure of the polypeptide that is folded into a compact stable conformation. Folding occurs as a stepwise process. Only the final form is ‘biologically active.’ Tertiary Structure: Combination of covalent and non- covalent interactions holding the structure together Tertiary Structure: Combination of covalent and non- covalent interactions holding the structure together Random coil Theoretical tertiary protein structure -helix May consist of -helices, -sheets, -turns and random coils. -sheet -turn Tertiary Structure: Protein Folding Protein folding occurs in a stepwise process. The structure is stabilised by non-covalent interactions (hydrogen bonds, ionic interactions, hydrophobic interactions), and the disulfide bond (only covalent bond). Only the final form is ‘biologically active.’ Solve Puzzles for Science: Foldit http://fold.it/portal/  Tertiary Structure: Protein Folding In aqueous environments, water-soluble proteins have an internal core that is hydrophobic, and external surfaces that are mainly hydrophilic. H2O Hydrophobic H2O portions are H2O folded inwards DENV2 Envelope protein H2O H2O Hydrophilic H2O H2O portions are H2O folded outwards Source: Modis et al., (2003) A ligand-binding pocket in the dengue virus envelope glycoprotein. PNAS 100:6986-6991. Tertiary Structure: Protein Folding Membrane proteins contain hydrophobic segments that help to embed them into the hydrophobic portions of the phospholipid bilayer. Hydrophilic G-protein coupled receptor contains Hydrophobic 7 transmembrane hydrophobic Hydrophilic segments. Source: Molecular biology of the cell. 4th edition. Exercise Let us suppose you are a PhD student in a virology lab. Given that this is the tertiary structure of the protein coat of dengue virus. DENV type 2 Envelope Protein Domain III Can you find the: α-helix? β-strand? β-turn? random coil? Domain I Domain II Picture Source: (Conference Proceedings) Tan, L.C.M. and Ng M.L. (2008). Development and characterization of dengue virus serotypes 1 to 4 recombinant envelope Domain III proteins and antibodies as diagnostic reagents for a biotin-streptavidin enhanced indirect ELISA. 5th International Meeting Bioprocess Technology: Asia Pacific, Singapore. Exercise FYI Exercises you can try out on your own. This protein has a tertiary structure. It contains 1 or more secondary structure. Locate as many secondary structures as you can. Human Serum Albumin Source: http://www.rcsb.org/pdb/explore/explore.do?structureId=1AO6 Summary on Protein Folding: Protein folding is dependent on types of amino acids present in the primary protein structure. The protein is held together by covalent peptide bonds. The peptide bonds interact with each other (via H-bond), giving rise to secondary structures, predominantly, α-helix and β- strands. The R-groups on every amino acid contribute to non-covalent interaction: H-bond, ιonic interaction, hydrophobic interaction. Cysteines residues contain τhiol functional group that can form covalent disulfide bonds with other cysteine residues. Quarternary Structures Can two or more proteins with tertiary structures combine to form a larger protein? E.g.: + =? Quarternary Structures YES! When 2 or more polypeptides (each with its own tertiary structure) combine together, they form a quarternary structure. E.g. Dengue virus envelope protein dimer. Source: Modis (2004) Structure of the dengue virus envelope protein after membrane fusion. Nature 427: 313-319.  Each individual polypeptide in the quarternary structure is known as a subunit. The polypeptide subunits are held together by non-covalent interactions and/or disulfide bonds (covalent), similar to the interactions that stabilize the internal structure of a protein. 1 Subunit 1 Subunit E.g. Dengue virus envelope protein dimer Source: Modis (2004) Structure of the dengue virus envelope protein after membrane fusion. Nature 427: 313-319. Quarternary Structures: Nomenclature Homo-dimer (similar protein subunits) Hetero-dimer Dimer (different types of protein subunits) Homo-trimer (similar protein subunits) Hetero-trimer Trimer (different types of protein subunits) Classical example of a protein with a quarternary structure: Haemoglobin File:1GZX Haemoglobin.png Haemoglobin is responsible for carrying oxygen around β α the body. Haemoglobin is found in red blood cells. The haemoglobin is made up of 4 subunits (2 alpha subunits and 2 beta α subunits) - heterotetramer β