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Foundations of Biochemistry: Amino Acids Protein Building Blocks Cindy McKinney, Ph.D. Reading Assignment • Chapter 6, Lieberman and Marks Lecture Objectives • Identify the basic structural properties of an amino acid, and identify the side chains of the 20 amino acids commonly found in proteins. • Predict the ionization of an amino acid at pH 7.0, including the side chain if it is an ionizable group (side chain pKa values will be provided where appropriate). • Recognize the nine essential amino acids and define why they are “essential.” • Define disulfide formation between two free cysteine side chains, and predict how disulfide formation affects protein primary structure. • Compare and contrast the chemical properties of each of the four weak forces: hydrogen bonds, dipole-dipole interactions, hydrophobic interaction, ionic interactions. • Define “biomolecular recognition” and relate how the four week forces govern the interaction of biomolecules. Proteins are made from Amino Acids Primary Protein Structure Linked aa chains (S-S bridge) of two polypeptides Why should doctors care about amino acids? Clinical relevance? Disease Examples (aa defects): • HOMOCYSTINURIA: error in methionine metabolismMAPLE SYRUP URINE DISEASE: inability to metabolize branched chain amino acids • SICKLE CELL ANEMIA: a single non-conservative amino acid (aa) mutation in hemoglobin (Hb) affects hemoglobin structure and function • CYSTIC FIBROSIS: most common aa mutation is Δ508F making a defective channel protein Therapeutic Example (mechanism of drug action): • PHARMACOLOGY: many drugs work by reacting with key amino acid side chains in enzymes and proteins Amino acids are the basic structural unit of all proteins • There are 20 common amino acids; 8 are essential A 'free' amino acid (a single amino acid) always has: -an amino group -NH2, -a carboxyl group -COOH -a hydrogen -H -a chemical group or side chain -"R” These are all joined to a central α-carbon atom (see following diagram) When used as the building blocks for polypeptide chains (protein) they will be linked to each other through the NH2 and COOH groups (shown later). The Basics: Structure of an Amino Acid α carbon Note: • There are 20 common amino acids used in proteins • Protonated amino group (physiological pH) • C-terminal carboxyl group • R-side chain from alpha carbon---yields distinct molecules because R can be: Non-polar, aromatic uncharged polar, sulfur containing, charged (basic or acidic) Peptide bond Classification of AA side chains-1 • Non-polar (aliphatic) “R” groups—hydrophobic (do not like contact with water/aqueous solutions; because of their aversion to water are frequently found in the interior (core) of a protein where they are “protected” from water Glycine (Gly; G) is the simplest and smallest of all amino acids, and the only one which is not optically active since it has a single hydrogen (H) atom as it's R side chain. Alanine (Ala; A) has a methyl group as it's R side chain. Valine (Val; V) has a slightly longer R side chain --there is a branch. Note: As the aliphatic side chains get longer they are also more hydrophobic Leucine (Leu; L) is very similar to valine except it has another methyl group attached to the R side chain Isoleucine (Ile; I) similar to leucine & valine except that the orientation of the atoms in the R side chain is slightly different. Isoleucine also has two centers of asymmetry. Proline (Pro; P) different from the other amino acids--- the R side chain is a 5 member ring derived from bonding to the α-carbon and the amino group. This structure effects the architecture of proteins. Considered aliphatic, it does not “mind” being in contact with water as much as the others. Classification of AA side chains-2 Structures of aliphatic amino acids: R R R α α R α R α α R=ring α proline Classification of AA side chains-3 Aromatic Amino Acids: the R group is an aromatic ring; aromatic rings are hydrophobic Phenylalanine (Phe; F) It contains a phenyl ring attached to a methylene group; the phenyl ring makes F a hydrophobic aa. The rings can stack on each other in a protein. Tyrosine (Tyr; Y) contains a hydroxyl group at the end of the phenyl ring--- it can form H-bonds. Tyrosine is less hydrophobic than phenylalanine. Tryptophan (Trp; W) has an indole ring attached to the methylene group. The indole ring is highly hydrophobic. Phenylalanine H-bonds Tyrosine Classification of AA side chains-4 Sulfur containing Amino Acids Cysteine (Cys; C) contains a sulphydryl group (-SH). This is extremely reactive, and can form hydrogen bonds. Cysteine --- very important to protein structure because it can form disulphide bridges (S-S) the -SH group of cysteine can form hydrogen bonds however, the aliphatic part of the side chain makes it quite hydrophobic. Methionine (Met; M) is a special amino acid it is the "start" amino acid in the process of translation (protein synthesis), and therefore, begins every single protein made. It also has a sulphur atom, this time it is in a thio-ether linkage, that can not form S-S bonds. Methionine has a highly hydrophobic R side-chain. Cysteine Methionine Classification of AA side chains-5 Hyrophillic Amino Acids—these aa “like” water but can be neutral, acidic or basic at physiological pH • Charged Negative Amino Acids (Acidic)—highly polar and negatively charged Aspartate (Asp; D) is really aspartic acid. It is called aspartate because it is usually negatively charged at physiological pH ---it is named for the carboxylate anion. (Compare acetic acid and acetate.) Glutamate (Glu; E) is also called glutamic acid. The R side chain of glutamate also has a carboxylate group which has a negative charge at physiological pH Classification of AA side chains-6 • Basic Amino Acids—R side chain (+) at physiological pH Lysine (Lys; K) Has long side chin although the side chain appears to be a hydrophobic hydrocarbon chain--- it is very polar because of the terminal amino group…therefore classified as a hydrophillic amino acid. Arginine (Arg; R) has the largest R side chains. The guanidino group attached to the side chain it has a high pKa value thus positively charged at physiological pH Histidine (His; H) contains an imidazole ring…this often sits inside the active site of an enzyme and helps bonds to be made or broken as the enzyme works (because it can exist in two states -uncharged or positively charged) Arginine Histidine Lysine Classification of AA side chains-7 • Neutral Amino Acids---not charged at physiological pH BUT all contain polar R groups that can form H-bonds (classified as hydrophillic) Serine (Ser; S) contains an aliphatic chain with a hydroxyl group---a hydroxylated the hydroxylated version of alanine. The -OH group makes the aa highly reactive and hydrophillic (readily forms hydrogen bonds) Threonine (Thr; T) another neutral amino acid that contains a highly reactive (and highly hydrophillic) –OH group. This is an aa that contains two centers of asymmetry (two asymetric carbon atoms) (also found in Ile). Asparagine (Asn; N) is the amide derivative of Aspartic acid. When the carboxylate side chain is amidated the resulting amide is uncharged. Note terminal amide group on N as opposed to the carboxyl group on aspartat Glutamine (Gln; Q) similar to Asparagine containing a terminal amide instead of a carboxyl group as in glutamate. These two are called the amide derivatives of their parent amino acids. amide Amino Acids---Characteristics • Each aa has a non-polar side chain that does not gain or lose protons or participate in H-bond formation • “R” side chain is best characterized as “oily” or lipid-like that promotes hydrophobic (excludes water) interactions. • Non-polar aa in proteins: In aqueous solutions or a polar environment, the hydrophobic side chains cluster together in the interior of the protein (see right hand figure) • Proline: Differs from other aa in that the R side chain and α-amino N form a rigid , five-membered ring structure. The unique geometry of proline contributes to the fibrous structure of collagen. Amino Acids---Characteristics-2 • • • • • • The amino acids with uncharged polar side chains have zero net charge at neutral pH Serine, threonine, and tyrosine each contain a polar hydroxyl (-OH) group that may be involved in H-bond formation Side chains of the polar OH group of serine, threonine, and rarely tyrosine can serve as an attachment site for phosphate (PO4) group The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can participate in hydrogen bond formation. Disulfide bonds: The –SH side chain of cysteine is a component of many enzyme active sites In proteins, the –SH groups of two cysteines can become oxidized to create a dimer (cystine) that contains a covalent cross-link or disulfide bond (-S-S-). Amino Acids---Characteristics-3 • Aspartic and glutamic acid are proton donors • At physiologic pH (7.4), the side chains of these amino acids are fully ionized, containing a negatively charged carboxyl (–COO-) group. They are therefore called aspartate or glutamate to emphasize these acids as being negatively charged at physiological pH. Amino Acids---Characteristics-4 • The R groups of Histidine, Lysine and Arginine are proton (H+) acceptors • At physiological pH the side chains of lysine and arginine are fully ionized and positively charged. • Histidine is weakly basic – When incorporated into a protein, its R group can be either positively charged or neutral depending on the ionic environment provided by the polypeptide chain – This property of histidine contributes to its role in the function of proteins such as hemoglobin Titration of an amino acid with a non-ionizable side chain Glycine Titration Curve Isoelectric point (pI)=no net charge Two pKas noticeable on graph: pKa1= -COOH group and pKa2= -NH3 Titration of an amino acid with a ionizable side chain Titration Curve of Histidine There are three pKas available: the α-COOH, the α-NH3 and the R side group (indole ring) This pKa depends on the R group structure Amino Acids Interactions Peptide A Peptide B • the hydrophobic effect, non-polar side chains will gather together in the protein interior whenever possible. • Aromatic side chains rarely exposed to polar / aqueous environment Amino Acids Interactions-2 Hydrogen Bonds/Electrostatic Interactions Peptide A Peptide B • Electronegative atoms (e.g., O, N) will attract electropositive H+ atoms This not a covalent bond, but an electrostatic interaction between atoms Amino Acids Interactions-3 Electrostatic Interactions Peptide A atoms with a formal positive or negative charge will attract one another Again, not a covalent bond, but an electrostatic interaction between oppositely charged atoms Peptide B Amino Acids Interactions-4 Disulfide Bridges: cysteine + cysteine • Cysteine may exist as a free sulfydryl (-SH) group or it may form a covalent disulfide (S-S) bond • Disulfide bonds play a key role in protein structure and function – they hold different parts of a protein together (ex: insulin) H+ removed • Disulfide bonds are sometimes considered part of the primary structure of proteins, but they contribute to secondary/tertiary structure (by holding polypeptide chain in proximity) • Disulfide bond formation: enzymatic process carried out when proteins are made. It is carefully regulated. Amino Acids Interactions-5 Ionizable R side chains play a significant role in protein structure and function Note: when solution pH=pKa, the side chain is 50% ionized Weak Forces: Understanding Protein Structure and Function Four weak forces at play: 1) van der waals 2) Hydrogen bonds 3) Ionic interactions/bonds 4) Hydrophobic interactions (water excluding) van der Waals Interactions • induced electrical interactions between closely approaching atoms/ molecules as their negatively-charged electron clouds fluctuate instantaneously in time •depends on the distance between interacting atoms • each interaction provides about 0.4 to 4 kJ of energy/ molecule • many vdw interactions in a system provides a large energy potential Hydrogen Bonds Examples between water molecules and serine residues in a protein Ionic Bonds Charged based attraction Hydrophobic Interactions H2O becomes organized • Tendency of water or polar molecules to exclude nonpolar groups or molecules • Interacting hydrophobic molecules have vdw interactions, but not a primary energetic / thermodynamic consideration • Entropy-driven process: water is more organized when it surrounds non-polar molecules • Drives the creation and maintenance of macromolecular structures: formation of lipid bilayers outside inside - Protein: Primary Structure • Primary structure of a protein = linear sequence of amino acids linked by peptide bonds Note: water loss during peptide bond formation What would a tri-peptide look like ? Variations in Primary Structure Amino acid linear sequence (N-terminal to C-terminal linked through peptide bonds) affect the secondary and tertiary structure. -protein structure even with the same sequence can vary among species -protein structure can also vary between >tissues (isoforms) > stage of development (fetal Hb vs. adult Hb) > individuals These changes are tolerated if: > confined to non-critical regions (variant) of the protein (not active enzyme sites) > conservative substitutions of one amino acid for another of similar structure > confer an advantage to protein function Polymorphisms in Protein Structure Variations can arise by mutations in DNA (point mutations, indels) may be an obvious dysfunction or disease A single point mutation in an invariant protein region has dire consequences when these changes occur frequently in a population =polymorphism Currently about 33% of loci in human genome appear to be Polymorphic sickle cell point mutation is a stable polymorphism in population > heterozygote provides some resistance to malaria Protein Families and SuperFamilies Divergent evolution: gene duplications are often associated with an ancestral gene; one protein may retain the original function while the duplicated protein may derive a new or related function Example: globin family (different proteins/function) Paralogs: myoglobin,α-globin, β-globin, γ-globin, δ-globin A model for the evolution of β-globin genes in mammals based on Phylogeny. The gene tree is drawn within the constraints of a species tree (21). The ancient gene duplication event (indicated by an arrow) gave rise to two ancestral genes, A and B. A was the progenitor of marsupial ω-globin and the β-like globin genes of birds. B gave rise to the β-like globin genes of mammals. Genes or pseudogenes that may be expected to occur are indicated by ?. To simplify the diagram, not all of the avian β-like globin genes (as exemplified by the chicken) are shown. PNAS 98: 1101-1106 Posttranslational Modification in Proteins A few amino acids may be modified after translation from the mRNA is completed---result of enzymatic activity on specific amino acids ≥ 100 different post translation modifications recognized These modifications may serve: 1) regulatory role 2) target/anchor a protein in a membrane 3) target a protein for degradation 4) enhance a protein’s interaction with another protein Posttranslational Modification in Proteins Types of posttranslational modifications (textbook figure 6.13) 1) Glycosylation: modification of amino acids at O-links or N-links N-linked small chain carbohydrates found in cell surface proteins protect cell from proteolysis or immune attack. 2) Fatty Acylation or Prenylation: many membrane proteins contain linked lipid groups that interact with lipids in membranes. 3) Regulatory Modifications: ADP-ribosylation, Phosphorylation, or Acetylation in many cases used to regulation activity of enzymatic proteins 4) Modifications of R group chains: blood clotting COOH added at γC of glutamate 5) Selenocysteine: required in a few enzymes for activity HSe-CH2-CH-COONH3+ Question • A glutamate is substituted for a valine in Hb causing sickle cell anemia. This is a nonconservative replacement. However, the substitution of an aspartate for a glutamate is a conservative replacement. How would you define non-conservative and conservative? What effect do you think this nonconservative replacement might have on protein structure?