Amino Acids, Peptides and Proteins PDF

Amino acids, peptides and proteins Marc A. Ilies, Ph. D. Lehninger - Chapter 3 (p75-114) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas or recitatio...

Amino acids, peptides and proteins Marc A. Ilies, Ph. D. Lehninger - Chapter 3 (p75-114) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas or recitation session ©MAIlies2024 1  Amino Acids: Building Blocks of Protein Proteins are linear heteropolymers of -amino acids Proteins mediate virtually every process that takes place in a cell Amino acids have properties that are well-suited to carry out a variety of biological functions – Capacity to polymerize – Useful acid-base properties – Diverse physical properties – Diverse chemical functionality 2  Most -amino acids share common structural features and are chiral The -carbon always has four substituents and is tetrahedral All amino acids (except proline) have: – an acidic carboxyl group – a basic amino group – an -hydrogen connected to the -carbon - a fourth substituent (R), unique – In glycine, the fourth substituent is also hydrogen – R group can be quite complex; nomenclature: 3  Proteins only contain L amino acids - the two stereoisomers (enantiomers) of alanine: (if relationship object/mirror image of object = enantiomers) L/D represent the relative configuration of amino acids to L- and D-glyceraldehyde: - L/D represent the configuration of the four substituents around the chiral carbon, and are not related with the sense of rotation of polarized light! 4 MW 5 ► Average MW of all 20 AAs is about 138 BUT there is a greater prevalence of low MW AAs in most proteins so the average MW of protein AAs is closer to 128. If we subtract 18 for water released upon formation of peptide bond, the average MW an amino acid residue = 110. Thus if we know the MW of a protein we can divide by 110 to get the approximate number of amino acids in that particular case. 6 Amino acids can be classified by R group 7  Amino acids can be classified by R group (either non-polar (Phe) or amphiphilic (Tyr, Trp) ► These amino acid side chains absorb UV light at 270–280 nm due to delocalized π electrons in the aromatic rings: 8  Amino acids can be classified by R group ► Cysteine can form disulfide bonds. ► These amino acids side chains can form hydrogen bonds 9 Amino acids can be classified by R group (polar, basic side chains, usually protonated at physiologic pH = 7.4) - the R group contains: 10 Amino acids can be classified by R group (polar, acidic side chains, usually fully ionized and negatively charged at physiologic pH = 7.4) 11  Uncommon amino acids found in proteins In the structure of prothrombin (blood- clothing protein), Ca2+ binding proteins In the structure of collagen (fibrous protein in connective tissue) In the structure of In the structure of myosin (contractile elastin (fibrous protein of muscles) protein in connective tissue) In the structure of some proteins One less methylene group compared to lysine! Found in Urea Cycle 12 Other important bio-active compounds derived from amino acids H CH2 C COOH CO2 NH2 CH2 CH2 NH2 HO N HO H N Serotonin Monoamine 5-Hydroxytryptophan H 5-HT neurotransmitter, HO (CNS, GI tract) a b g H HOOC CH2 CH2 CH2 NH2 COOH HO g-Aminobutyric Acid ( GABA) NH2 Catecholamine Precursor to main inhibitory Dihydroxy Phenylalanine catecholamines neurotransmitter DOPA (dopamine, in CNS I I norepinephrine, H epinephrine) HO O CH2 C COOH NH2 I I Thyroxine T4 3,5, Diiodotyrosine Precursor for Triiodothyronine T3 (thyroid hormone) 13  Ionization of Amino Acids At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form. At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions. At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form. Net Charge = 0 Not common in aqueous solution 14  Amphoteric Nature of Amino Acids H H R C COO + proton donor R C COO + H Acid NH NH2 3 Zwitterion H H + R C COO + H R C COOH proton acceptor Base NH 3 NH 3 diprotic acid + H + H H H H R C COOH R C COO R C COO NH NH NH2 3 3 Net Charge +1 0 -1 15  Amino acids can act as buffers in solution Amino acids with uncharged side chains, such as glycine, have two pKa values: -the pKa of the -carboxyl group is 2.34 Cation  Zwitterion  Anion -the pKa of the -amino group is 9.6 It can act as a buffer in two pH regimes: Buffer Regions 16  Amino acids carry a net charge of zero at a specific pH (the pI) Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups For amino acids without ionizable side chains, the Isoelectric Point (equivalence point, pI) is pK1 + pK2 pI = 2 At this point, the net charge is zero – AA is least soluble in water – AA does not migrate in electric field Chemical environment in amino acids affects their pKa values Due to simultaneous presence in amino acid structure of both COOH and NH2 groups: - -carboxy group is much more acidic than in carboxylic acids - -amino group is slightly less basic than in amines 18  Ionizable side chains can show up in titration curves Ionizable side chains can be also titrated Titration curves are now more complex pKa values are discernable if two pKa values are more than two pH units apart Zwitter Ion Zwitter Ion pI=3.22 Monoamino monocarboxylic AA Monoamino dicarboxylic AA  Ionizable side chains can show up in titration curves Ionizable side chains can be also titrated Titration curves are now more complex pKa values are discernable if two pKa values are more than two pH units apart pK 1 pK 2 pK R Zwitter Ion Lysine R pI= 9.75 Diamino monocarboxylic AA Histidine 20 General calculation of the pI when the side chain is ionizable Identify species that carries a net zero charge Identify pKa value that defines Zwitter Ion the acid strength of this zwitterion Identify pKa value that defines the base strength of this pI=3.22 zwitterion Take the average of these two pKa values  Formation of Peptides and Proteins Peptides are small condensation products of amino acids They are “small” compared to proteins, which have Mw > 10 kDa Equilibrium lies on the side of hydrolysis thus energy is need to synthesize peptides BUT hydrolysis rate is slow (kinetically stable). In the absence of a catalyst a peptide bond in an aqueous environment would last > 1,000 years !! 22  Peptide nomenclature Numbering (and naming) starts from the amino terminus: AA1 AA2 AA3 AA4 AA5 H 2N-Ser-Gly-Tyr-Ala-Leu-COOH 23  Ionization of Peptides H2N-Ala-Glu-Gly-Lys-COOH - In a typical peptide the terminal amino, carboxyl group contribute to the pI, as well as, the ionizable R groups: - Peptides and proteins have characteristic pI values 24  Conjugated proteins ► Some proteins contain other chemical compounds besides amino acids: cofactors  functional non-amino acid component  e.g. metal ions or organic molecules (coenzymes) coenzymes  organic cofactors  e.g. NAD+ in lactate dehydrogenase prosthetic groups  covalently attached cofactors  e.g. heme in myoglobin other modifications  TABLE 3-4 Conjugated Proteins Class Prosthetic group Example Lipoproteins Lipids β1-Lipoprotein of blood Glycoproteins Carbohydrates Immunoglobulin G Phosphoproteins Phosphate groups Casein of milk Hemoproteins Heme (iron porphyrin) Hemoglobin Flavoproteins Flavin nucleotides Succinate dehydrogenase Metalloproteins Iron Ferritin Zinc Alcohol dehydrogenase Calcium Calmodulin Molybdenum Dinitrogenase Copper Plastocyanin 26  Separation and purification of proteins Source of proteins diverse (tissue, individual cells) → lysis to generate a crude extract Separation of proteins from crude extracts relies on differences in physical and chemical properties: – Charge – Size – Affinity for a ligand – Solubility – Hydrophobicity – Thermal stability Chromatography is commonly used for preparative separation 27 Electrophoresis for Amino Acid or Protein Analysis Separation in analytical scale is commonly done by electrophoresis – Electric field pulls amino acids or proteins according to their charge – Gel matrix hinders mobility of proteins according to their size and shape pI = 9.7 + pH = 7 An amino acid placed in an environment with a pH below its pI will have a net (+) charge and move toward the negative electrode (cathode), ie pI = 9.7 in a pH of 7.0. (At a pH of 9.7, this amino acid will not migrate toward either electrode, while at pH of 13 it will have a net (-) charge) A Rule to Remember !!!: if working pH > pI, then protein or amino acid charge is negatively charged if working pH < pI, then protein or amino acid charge is positively charged 28 Electrophoresis for Amino Acid or Protein Analysis - Samples loaded onto the gel matrix (polyacrylamide), together with MW markers, controls (purified proteins) - Electric field applied - Gel isolated, stained with dye 29  Protein Structure A description of all covalent bonds, Particular stable The arrangement including disulfide arrangements of in space of a bonds. Basically amino acid residues Describes all of protein which the amino acid resulting in recurring the 3-D polypeptide contains 2 or sequence. patterns folding more polypeptide units 30  Goals and Objectives Upon completion of this lecture at minimum you should be able to answer the following: ► Structure, properties and naming of amino acids ► Structure and properties of peptides and proteins ► Ionization behavior of amino acids and peptides, and the isoelectric point ► Classes of conjugated proteins, cofactors, coenzymes, prosthetic groups ► Methods to separate and purify peptides and proteins, electrophoresis of proteins, behavior in electric field 31