Biochemistry I Lecture 1: Amino Acids and Peptides (PHBC 522) PDF

Biochemistry I PHBC 522 Hans-Georg Breitinger Mohamed Z. Gad Sahar Mohamed Sally Ibrahim Ingy Hashad Ulrike Breitinger Raghda Elsabbagh Marina Fam Nancy Turky Noura...

Biochemistry I PHBC 522 Hans-Georg Breitinger Mohamed Z. Gad Sahar Mohamed Sally Ibrahim Ingy Hashad Ulrike Breitinger Raghda Elsabbagh Marina Fam Nancy Turky Noura Ayman Christine Adel Heba Nafea Zeina Ibrahim Omnia Diaa Alaa Mohamed Faculty for Pharmacy and Biotechnology Lecture 1 Course Outline Amino Acids and Peptides PHBC 522 Lecture 1 1  Lecture 1 Amino Acids and Peptides PHBC 522 Lecture 1 2  Lecture Contents and Learning Outcomes At the end of this lecture, you should be able to … - Review of the basic concepts of Chemistry, Biology, Thermodynamics that pertain to Biochemistry - Recall functional groups and non-covalent interactions relevant to Biochemistry - Name and draw the structures of the 20 proteinogenic amino acids - Describe posttranslational modifications of amino acid side chains in a protein and their relevance. - Describe formation of a peptide bond, its thermodynamic properties, conformations (partial double bond, cis/trans), and the arrangements of amino acids in a peptide chain - Describe the rotational conformation about the F and Y bonds in a (poly)peptide, how they are collected in the Ramachandran plot, and their relevance to protein conformation and structure Reference: Stryer/Berg/Tymoczco 5th edition - 1.3. Chemical Bonds in Biochemistry - 2.1. Key Organic Molecules Are Used by Living Systems - Chapter 3. Protein Structure and Function - 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids - 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains - 3.2.1. Proteins Have Unique Amino Acid Sequences That Are Specified by Genes - 3.2.2. Polypeptide Chains Are Flexible Yet Conformationally Restricted 3 PHBC 522 Lecture 1  Basic Chemistry in Biochemistry Chemistry Elements, atoms, electrons, … Covalent bonds, ionic bonds, non-covalent interactions Acids/Bases, acidity, buffers Organic Chemistry Functional groups and their properties (to be revised) Basic Reaction mechanisms Reactivities, covalent bonds, structures, conformation “one needs as much Organic Chemistry for Biochemistry as for Organic Chemistry” Physical Chemistry Thermodynamics, Kinetics Biology Basic concepts of life, cells, biomolecules, … PHBC 522 Lecture 1 4  Organic Chemistry in Biochemistry – Functional Groups PHBC 522 Lecture 1 5 https://sites.google.com/site/csetstudyguidechemistry/home/6-1b-organic-chemistry-biochemistry---various-organic- functional-group  Organic Chemistry in Biochemistry – Functional Groups Alanine Phenylalanine Leucine Tyrosine Isoleucine Serine Threonine Lysine Tyrosine Aspartate Glutamate Asparagine Cysteine Glutamine Cystine (dimer) Methionine Histidine Arginine Phospho– serine threonine tyrosine PHBC 522 Lecture 1 6 https://courses.ecampus.oregonstate.edu/bb450/lecture/funct.pdf Non-covalent reversible forces between biological molecules 1 1) Hydrogen bond formed between lone electron pairs on O, N, F and an H atom (bearing a partial positive charge in a polar covalent bond). Requires polar bonds, not formed between hydrocarbons. PHBC 522 Lecture 1 7  Non-covalent forces between biological molecules 2 2) Electrostatic interaction (salt bridges) q1 q 2 q = charge ; r = distance D = dielectric constant E 2 Water: D = 80 Lipids: D = 2 Dr Dipole – dipole interaction PHBC 522 Lecture 1 8  Non-covalent forces between biological molecules 3 3) Van der Waals interaction (London forces): Electrons fluctuate around the nucleus  Temporary polarization of the of the electron clouds  temporary dipoles  These temporary dipoles can induce temporary dipoles in neighbouring molecules  attraction  Temporary dipoles (and thus attraction) are very short-lived This is the only attractive force between non-polar molecules PHBC 522 Lecture 1 9  Non-covalent reversible forces Holds the two strands of the DNA double helix together (hydrogen bonds) Folds polypeptides into such secondary structures as the alpha helix and the beta conformation Enables enzymes to bind to their substrate Enables antibodies to bind to their antigen Enables transcription factors to bind to each other Enables transcription factors to bind to DNA Enables proteins (e.g. some hormones) to bind to their receptor Permits the assembly of such macromolecular machinery as – ribosomes – actin filaments – microtubules PHBC 522 Lecture 1 10  The Laws of Thermodynamics First Law of Thermodynamics: The energy of the universe (hence of any system and its surroundings) is constant. Energy can never be generated nor destroyed, only converted. Second Law of Thermodynamics: The Total entropy of a system and its surroundings always increases for spontaneous processes Entropy: measure for ‘disorder‘, randomness, number of possible arrangement of molecules in space. Entropy can decrease locally only if entropy increases by an equal or great amount of other parts in the universe. PHBC 522 Lecture 1 11  Thermodynamics in Biochemistry – practical effects Hydrophobic effect Weak interactions of water molecules with nonpolar molecules increase the order of those water molecules forming the solvation shell (= decrease in entropy). When nonpolar molecules aggregate, fewer water molecules are needed for solvation (volume ~ r3, surface ~ r2)  overall entropy of water increases. The hydrophobic effect favours aggregation of nonpolar groups in proteins, compensating loss of entropy in protein folding (generation of highly structured species). PHBC 522 Lecture 1 12 Amino Acids and Proteins PHBC 522 Lecture 1 13  Proteins – the most versatile macromolecules of life Proteins - Represent 50% of the dry weight of E.Coli cell - Are crucial for almost all biological processes - Catalysts (enzymes) - Storage molecules (e.g. oxygen) - Structural molcules (keratin, collagen) Proteins - made of monomeric units – amino acids - contain a wide range of functional groups (OH, SH, NH2, COOH, CONH2, aliphatic, aromatic, - variable contact surfaces – can interact with many molecules, and other proteins - variable structures: rigid or flexible, often both found in one protein PHBC 522 Lecture 1 14  Amino Acids – building blocks of proteins Amino acids contain a carboxyl (=acid) group -COOH, and an amino group –NH2. ANY compound that contains these two functional groups is called an amino acid, regardless where the functional groups are located. Nomenclature: the –CH2– group next to the –COOH is called a, the following one b, etc going through the greek alphabet. Proteins are made of a-amino acids PHBC 522 Lecture 1 15  Proteinogenic amino acids – structural principle 19 out of 20 proteinogenic amino acids are chiral, ie they can exist as image and mirror image. However, in nature only one isomer is found, all natural proteins are made of L-amino acids. If you figure the amino acid as a bridge from –COOH to the –NH2 group, the side chain is always on the left. (Not quite the IUPAC definition, but it will do here) O H2N – CH – COOH | H2N CH C OH is common to all proteinogenic amino acids, side chain R varies R PHBC 522 Lecture 1 16  Amino acids exist as zwitterons (charge = 0) -COOH: pKA ~ 2 -NH2: pKA ~ 9 PHBC 522 Lecture 1 17  The 20 proteinogenic amino acids 1 1) Aliphatic side chain: hydrophobic effect  dense packing inside protein Increasingly hydrophobic small, ‘versatile‘ achiral 2nd centre of chirality (always S) PHBC 522 Lecture 1 18  The 20 proteinogenic amino acids 2 2) Aromatic side chain: p-interactions Hydrophobic Hydrophilic, Bulky, phosphorylated moderately polar PHBC 522 Lecture 1 19  The 20 proteinogenic amino acids 3 3) Side chain containing S 4) Side chain containing OH 5) Proline OH: polar, phosphorylated Imino acid (unique) Sterically restrained SH: reactive Thioether, Induces kinks in Disulfide bulky protein chain (esp. PP) bonds PHBC 522 Lecture 1 20  The 20 proteinogenic amino acids 4 6) Basic O H2N CH C OH CH2 N NH O H2N CH C OH CH2 H N Basic, pKA 11 – 12 pKA ~ 6 N ‘proton shuttle‘ H PHBC 522 Lecture 1 21  The 20 proteinogenic amino acids 5 7) Acidic / Carboxamides Acidic, pKA ~ 4 Polar, H-bonds PHBC 522 Lecture 1 22  The 20 proteinogenic amino acids – finishing touches Posttranslational modifications, fine-tuning of residue chemistry Phosphorylated H-bonds, Ca2+ chelator major physiological collagen helix blood clotting on/off switch PHBC 522 Lecture 1 23  The 20 proteinogenic amino acids – finishing touches Hydroxyproline stabilizes collagen fibres, giving strength to - Connective tissue - skin - bones Lack of vitamin C  Proline is not hydroxylated  incorrectly assembled collagen  decay of connective tissue  Loss of teeth, hemorrhages, death  Scurvy Scurvy was feared among seamen, Help: citrus fruits, sour cabbage in diet PHBC 522 Lecture 1 24  The 20 proteinogenic amino acids – finishing touches Phosphorylation is the major on/off – switch for protein activity Proteins can be synthesized in the cell but are inactive. Upon phosphorylation, protein becomes active.  No need to go through all the steps of protein synthesis  Protein is present and only needs to be switched on or off  Works both ways: some proteins are active upon phosphorylation, some are activated by dephosphorylation  We have a large number of enzymes catalyzing these reactions: kinases and phosphatases PHBC 522 Lecture 1 25  The 20 proteinogenic amino acids – finishing touches Cysteine can form disulfide bridges , these stabilize protein structures PHBC 522 Lecture 1 26  The 20 proteinogenic amino acids – finishing touches Disulfide bridges can crosslink proteins, defining and stabilizing their structure. The example below is for insulin, two chains are held together by disulfide bridges Disulfide bridges can be broken by mercaptoethanol. It contains a –SH group which is oxidised by the –S–S– group of the protein. PHBC 522 Lecture 1 27  The biogenic amino acids – pKA values pKA and pI Values of Polyfunctional Amino Acids α-CO2H α-NH3 Side Chain Amino Acid pI pKa1 pKa2 pKa3 Arginine 2.00 9.00 13.20 11.15 Aspartic Acid 2.01 9.82 3.83 2.80 Cysteine 1.71 10.78 8.33 5.02 Glutamic Acid 2.19 9.67 4.25 3.22 Histidine 1.82 9.17 6.04 7.59 Lysine 2.17 9.00 10.80 9.65 Tyrosine 2.20 9.11 10.07 5.66 ~2 ~9 PHBC 522 Lecture 1 28 The biogenic amino acids – names and abbreviations PHBC 522 Lecture 1 29  Amino acids – side chain properties Properties of Amino Acids Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr A R N D C E Q G H I L K M F P S T W Y acidic acyclic aliphatic aromatic basic buried charged cyclic hydrophobic large medium negative neutral polar positive small surface PHBC 522 Lecture 1 30  Amino acids – side chain properties Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr A R N D C E Q G H I L K M F P S T W Y acidic basic charged negative positive neutral polar hydrophobic acyclic cyclic aliphatic aromatic buried surface large medium small PHBC 522 Lecture 1 31  Amino acids can be linked by peptide bonds Peptide bond - condensation reaction (loss of one H2O) - Thermodynamically disfavoured: equilibrium lies on side of hydrolysis - Kinetically stable: peptide bond in aqueous solution ~ 1000 years - Several amino acids can be linked, forming a polypeptide chain - backbone: -NH2, a-Carbon, -COOH ; side chains protrude from backbone - Convention: amino terminus taken as beginning of polypeptide: N  C Side chains Peptide backbone Side chains PHBC 522 Lecture 1 32  Peptides – polarity N  C C-terminus N-terminus (carboxyl end) (amino end) PHBC 522 Lecture 1 33  The peptide bond is a rigid, planar unit The amide (peptide) bond exists in two resonance forms  Partial double bond character of the peptide C – N bond Bond lengths: 1.24 Å C–N single: 1.49 Å C=N double: 1.27 Å 1.46 Å C–N peptide: 1.32 Å 1.51 Å 1.32 Å PHBC 522 Lecture 1 34 Peptide bonds – rigid, planar structures PHBC 522 Lecture 1 35 The rigid peptide unit defines the protein backbone PHBC 522 Lecture 1 36  Peptide bonds may be cis or trans configured The planar arrangement of the atoms in the peptide bond (which is required for resonance) can be realised in two ways: Trans: Carbonyl-O and amide-H on different sides of the peptide group Cis: Carbonyl-O and amide-H on the same side of the peptide group Usually, the trans configuration is strongly favoured, since there is no steric hindrance O H C–N H C–N O cis trans 37 PHBC 522 Lecture 1  Proline: cis or trans configuration equally (un)stable Trans configuration is generally favored over cis: - Only 116 (0.36%) of 32,539 angles in 154 X-ray structures were found to be cis (Stewart et al. 1990). - However...... some specific bonds are often cis, such as - Tyr-Pro (25%), Ser-Pro (11%), X-Pro (6.5%) This leaves phi and psi for flexible folding of the chain. However, steric conflicts limit even these angles as well.  For proline, there is equal steric hindrance in the cis and trans configuration, so both are actually realised. Prolyl isomerase catalyses the conversion from cis to trans and vice versa. PHBC 522 Lecture 1 38 The rigid peptide unit defines the protein backbone F Y The rigid peptide bond allows only two degrees of rotational freedom: (B) about the N – a-C – bond: F (phi) (C) about the a-C – C(OO-) bond: Y (psi: C – C) PHBC 522 Lecture 1 39  Values for F and Y are restricted Steric clashes limit the the range of values that F and Y may take. Minimum Energy Conformation f = -160º. Potential Energy as a Function of f The x-axis runs from -180º to +180º. Maximum Energy Conformation f = +16º. PHBC 522 Lecture 1 40  Ramachandran plots indicate allowed regions for F and Y By plotting in a diagram the ‘allowed’ values of F and Y (so that steric clashes are avoided), one obtains a plot that shows the allowed regions for both angles. This is called the Ramachandran plot. Note that ca. 75 % of all possible conformations are excluded by steric constraints. PHBC 522 Lecture 1 41  Summary Biochemistry includes concepts and molecular approaches from General Chemistry, Organic Chemistry, and Physical Chemistry. Non-covalent interactions (H-bonds, electrostatic interactions, van der Waals forces) are critical forces that bring molecules and (bio)macromolecules together and help orient themselves. Proteins are made up of 20 amino acids. These have a constant, L-amino acids part and variable side chains (acidic, basic, aliphatic, aromatic, SH, OH, amines, amides, heterocycles). Amino acids condense, forming peptide bonds. This reaction is thermodynamically disfavoured, but the peptide bond is kinetically stable. Amino acids can be modified after protein synthesis to give particular properties to a protein. Modificaton include hydroxylation, carboxylation, phosphorylation, formation of disulphide bridges. The peptide bond has a partial C=N double bond, rendering it a planar, rigid unit. Rotation is possible about two bonds: N – a-C (angle F); a-C – C(=O) (Y). The Ramachandran plot displays allowed values for F and Y. Due to steric clashes, only ca. 25 % of all possible orientations can be adopted. PHBC 522 Lecture 1 42