Amino Acids Jana Novotna Department of Medical Chemistry and Clinical Biochemistry 2016
General characteristics of amino Building blocks of proteins. acids (AA) 20 common AA - encoded by standard genetic code, construct proteins in all species. Primary structure of AA determinates unique three-dimensional structure, function, binding sites of proteins for different interactions. Important intermediates in metabolism (porphyrins, purines, pyrimidines, creatin, urea etc). Some of AA have hormonal or catalytic function. Several genetic disorders are cause in amino acid metabolism errors (aminoaciduria - presence of amino acids in urine)
The basic structure of amino acids
Simple monoamino monocarboxyl a-amino acid - diprotic acid - can yield protons when fully protonated
AA have characteristic titration curve +1 +0.5 0-0.5-1 pk a COOH + pk a NH 3 + pi = 2 Proton donor Proton acceptor 2.34 + 9.60 pi = 2 = 5.97 + At the midpoint pk=9.60 there is equimolar concentration of proton donor and proton acceptor. Dipolar ion Isoelectric ph = pi + At the midpoint pk1=2.34 there is equimolar concentration of proton donor and proton acceptor. Fully protonated form at wery low ph Proton donor Proton acceptor Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Dissociation of the side chains of AA
Titration curve for (a) glutamate and (b) histidine (the pk a of the R group is designed here as pk R ) pi = pk 1 + pk R 2.19 + 4.25 = = 3.22 pi = pk R + pk 2 6.0 + 9.17 = = 7.59 2 2 2 2 The isoelectric points reflect the nature of ionizing groups present. Glutamate has two COO - groups, histidine has two groups (a-nh + 3 and imidazole group). Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Binding interactions of AA Electrostatic interactions A disulfide bond
The stereochemistry of AA Chiral molecules existing in two forms http://www.imb-jena.de/~rake/bioinformatics_web/gifs/amino_acids_chiral.gif
The two stereoisomers of alanine a-carbon is a chiral center Two stereoisomers are called enantiomers. The solid wedge-shaped bonds project out of the plane of paper, the dashed bonds behind it. The horizontal bonds project out of the plane of paper, the vertical bonds behind.
Classification based on chemical constitution Small amino acids Glycine, Alanine Branched amino acids Valine, Leucine, Isoleucine Hydroxy amino acids (-OH group) Serine, Threonine Sulfur amino acids Cysteine, Methionine Aromatic amino acids Phenylalanine, Tyrosine, Tryptophan Acidic amino acids and their derivatives Aspartate, Asparagine, Glutamate, Glutamine Basic amino acids Lysine, Arginine, Histidine Imino acid - Proline
Essential and nonessential AA Arginine* Histidine* Isoleucine Leucine Valine Lysine Methionine Threonine Phenylalanine Tryptophan Alanine Asparagine Aspartate Glutamate Glutamine Glycine Proline Serine Cysteine** (from Met) Tyrosine**(from Phe) * Essentials only for children ** Conditionally essentials
Uncommon amino acids found in proteins Intermediates of biosynthesis of arginin and in urea cycle
Peptide bond formation
Proteins
How a sequence of AA in a polypeptide chain is translated into a discrete, three dimensional protein structure? The three-dimensional structure is determined by amino acid sequence. The function depends on the structure. The most important forces stabilizing the specific structure are noncovalent interactions.
The peptide bond is rigid and planar The peptide bond is a hybride between the resonance forms the carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, partial double form of peptide bond itself. The N-Ca and Ca-C can rotate on angles f and j, resp., the peptide C-N bond is not free to rotate. Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Primary structure of proteins Knowledge of primary structure of protein is require for understanding of : the protein s structure the mechanism of protein action on molecular level the interrelationship with other proteins in evolution Sequencing of protein is an aids for : the study of protein modification the prediction of the similarity between two proteins The determination of the primary structure of a protein requires a purified protein. The cloning of the genes for many proteins and the sequencing of gene is a much faster method to obtain the amino acid sequence.
The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. the N-terminal end - on the left (the end bearing the residue with the free a-amino group) the C-terminal end - on the right (the end with the residue containing a free a-carboxyl group).
Knowledge of primary structure of insulin aids in understanding its synthesis and action. 1. Pancreas produces single chain precursor proinsulin 2. Proteolytic hydrolysis of 35 amino acid segment C peptide 3. The remainder is active insulin (two polypeptide chains A and B) covalently joined by disulfide bonds Amino acid identity in different animals: Human, hors, rat, pig, sheep, chicken insulin have differences only in residues 8, 9, and 10 of the A chain and residue 30 of the chain B
Higher levels of protein organization Secondary structure The second level of protein structure determined by attractive and repulsive forces among the amino acids in the chain. It is the specific geometric shape caused by intra-molecular and intermolecular hydrogen bonding of amide groups. Tertiary structure Three dimensional structure of polypeptide units (includes conformational relationships in space of side chains R of polypeptide chain). Quaternary structure Polypeptide subunits non-covalently interact and organize into multi-subunit protein (not all proteins have quaternary structure). The folding of the primary structure into secondary, tertiary and quaternary structure appears to occur in most cases spontaneously. Cystein disulfide bonds are made after folding
Protein secondary structure The a-helix a-helix - right-handed coiled conformation. Every backbone N-H group of peptide bond donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier. 3.6 amino acid residues are per 360 o turn. Region richer in Ala, Glu, Leu, Met, and poorer in Pro, Tyr, Ser tend to form a-helix. The formation of the a-helix is spontaneous.
b sheets 2 strands (segments) of polypeptide chains are stabilized by H-bonding between amide nitrogens and carbonyl carbons. Polypeptide segments are aligned in parallel or anti-parallel direction to its neighboring chains. b-structure gives plated sheet appearance side chain groups are projected above and below the plane generated by the hydrogen-bonded polypeptide chains.
b sheets In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C- terminal ends is the same). In anti-parallel sheets adjacent chains are aligned in opposite directions. The large number of hydrogen bonds maintain the structure in a stretched shape.
Protein tertiary structure The folding pattern of the secondary structural element into 3D conformation The tertiary structure of a protein Forces that give rise to tertiary structure b plated sheets Hydrophobic interaction a helical regions
Examples of the tertiary structure Examples of a,b-folded domains in which b-structural strands form a b barrel in the centre of the domain Examples of b- folded domains
Protein quaternary structure The arrangement of the protein subunit in the threedimensional complex constitutes quaternary structure. Hemoglobin Four subunits (two a and two b subunits) are associated in the quaternary structure
Forces controlling protein structure Hydrophobic interaction forces: Interaction inside polypeptide chains (amino acids contain either hydrophilic or hydrophobic R-groups). Interaction between the different R-groups of amino acids in polypeptide chains with the aqueous environment. A non-polar residues dissolved in water induces in the water solvent a solvation shell in which water molecules are highly ordered. Two non-polar groups in the solvation shell reduce surface area exposed to solvent and come very close come together. Hydrogen bonds: Proton donors and acceptors within and between polypeptide chains (backbone and the R-groups of the amino acids). H-bonding between polypeptide chains and surrounding aqueous medium.
Electrostatic forces: Charge-charge interactions between oppositely charged R-groups such as Lys or Arg (positively charged) and Asp or Glu (negatively charged). Ionized R-groups of amino acids with the dipole of the water molecule. van der Waals forces: Weak non-colvalent forces of great importance in protein structure, the sum of the attractive or repulsive forces between molecules Force is caused by the attraction between electron-rich regions of one molecule and electron-poor regions of another
Protein denaturation and folding Denaturation is a loss of the threedimensional. The protein loss of it function. Denaturation by heat has complex effect on the weak interactions (primarily by disrupting hydrogen bonds). Extremes of ph alter the net charges on the protein, causing electrostatic repulsion and the disruption of some hydrogen bonding. Organic solvents and detergents act primarily by disrupting hydrophobic interactions Renaturation is process in which protein regains its native structure Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Some proteins undergo assisted folding Not all proteins fold spontaneously and require molecular chaperons. Chaperons interact with partially or improperly folded polypeptides Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Protein misfolding Amyloid fibre is an insoluble extracellular formation (amyloidoses). They arise from at least 18 inappropriately folded versions of proteins and polypeptides present naturally in the body. Alzheimer s disease b-sheet undergoes partial folding, associates partially with the same region in another polypeptide chain (the nucleus of amyloid). Prion protein Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Protein structure 1. Globular proteins are compactly folded and coiled. 2. Fibrous proteins are more filamentous or elongated. 3. Peptides Small peptides (containing less than a couple of dozen amino acids) are called oligopeptides. Long peptides are called polypeptides. Peptides have a "polarity"; each peptide has only one free a-amino group (on the amino-terminal residue) and one free (non-side chain) carboxyl group (on the carboxy-terminal residue)
Functional roles of proteins 1. Dynamic function transport metabolic control contraction catalysis of chemical transformation 2. Structural function bone, connective tissue
Classification of proteins by bioloical function 1. Enzymes (lactate dehydrogenase, DNA polymerase) 2. Storage proteins (ferritin, cassein, ovalbumin) 3. Transport proteins (hemoglobin, myoglobin, serum albumin) 4. Contractile proteins (myosin, actin) 5. Hormones (insulin, growth hormone) 6. Protective proteins in blood (antibodies, complement, fibrinogen) 7. Structural proteins (collagen, elastin, glycoproteins)
Types of proteins Globular proteins Spheroid shape Variable molecular weight Relatively high water solubility Variety function roles catalysts, transporters, control proteins (for the regulation of metabolic pathways and gene expression) Fibrous proteins Rodlike shape Low solubility in the water Structural role in the organism Lipoproteins Complex of lipids with protein the addition of lipids, fatty acylation (palmitoylation) Glycoproteins Contain covalently bound carbohydrate O- or N-glycosylation
Globular proteins Globular proteins, such as most enzymes, usually consist of a combination of the two secondary structures. For example, hemoglobin is almost entirely alpha-helical, and antibodies are composed almost entirely of beta structures.
Fibrilar proteins Collagen Keratin
Lipoproteins Multicomponent complexes of protein and lipids. The lipids or their derivatives may be covalently or non-covalently bound to the proteins. Examples of lipoproteins: many enzymes, transporters, structural proteins, antigens, adhesins and toxins. The function of lipoprotein particles - transport of lipids (fats) and cholesterol around the body in the aqueous blood, in which they would normally dissolve
Glycoproteins Glycoproteins have covalently attached sugar molecules at one or multiple points along the polypeptide chain Glycoproteins are: hormones extracellular matrix proteins proteins involved in blood coagulation antibodies mucus secretion from epithelial cells protein localized on surface of cells receptors (transmit signals of hormones or growth factors from outside environment into the cell) Sugar molecules are: glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine
Structure-Function Relationship of Protein Families
Hemoglobin and myoglobin Human hemoglobin occurs in several forms. Consist of four polypeptide chains of two different primary structure. Bind oxygen in the lung and transport the oxygen in blood to the tissues and cells.
Myoglobin is a single polypeptide chain with one oxygen binding site. Binds and release oxygen in cytoplasm of muscle cells. Hemoglobin and myoglobin molecules each contain a heme prosthetic group. Protein without prosthetic group is designated as apoprotein. Complete protein is a holoprotein
Oxygen binding to Fe 2+ of heme in hemoglobin Proximal histidine binding pulls Fe 2+ above the plane of porphyrine ring O 2 binding cause conformational changes which pulls Fe 2+ back
Contractile elements of muscles Myosin thick filament of the muscle Actin thin filament of the muscle G-actin (globular actin) F-actin (fibrilar actin) Tropomyosin Troponin One of the biologically important properties of myosin is its ability to combine with actin to generate muscle contraction.
Biological membrane proteins Integral membrane proteins Peripheral membrane proteins Channels and pores Erythrocyte membrane Diagram of a voltage-sensitive sodium channel α-subunit. G - glycosylation, P- phosphorylation, S - ion selectivity, I - inactivation, positive (+) charges in S4 are important for transmembrane voltage sensing.
Membrane receptors 1. b-polypeptide stretch extendings from a-helix. 2. Seven membrane-spanning domains. 3. Recognize catecholamines, principally norepinephrine. Hormone activates receptor. Hormone-receptor mediated stimulation of intracellular signalling cascade. Proposed model for insertion of the b 2 adrenergic receptor in the cell membrane
DNA binding proteins Regulatory proteins binding to DNA sequence can promote either an activation or repression of the rate of gene transcription into mrna Helix-turn-helix binding proteins The zinc finger motif The leucine zipper motif The zinc finger motif Helix-turn-helix motif