Week 3-Amino acids-Peptides-Proteins - Short (1).pptx

AMINO ACIDS, PEPTIDES, AND PROTEINS Assist. Prof. Dr. Derya DİLEK KANÇAĞI Room Number: 511 E-mail: [email protected] Office Hour: Wednesday 13.00-15.00 Proteins mediate virtually every process that takes place in a cell, exhibiting an almost endless diversity of functions… • • • • • The most abundant biological macromolecules, Occur in great variety, Thousands of different kinds may be found in a single cell, The molecular instruments through which genetic information is expressed, Different properties and activities by joining a common set of 20 amino acids in many different combinations and sequences • Enzymes, hormones, antibodies, transporters, lightharvesting complexes in plants, the flagella of bacteria, muscle fibers, feathers, spider webs, rhinoceros horn, antibiotics, and myriad other substances that have distinct biological functions Central Principles in Biochemistry Principle 1 Principle 2 • In every living organism, proteins are constructed from a common set of 20 amino acids. • In proteins, amino acids are joined in characteristic linear sequences through a common amide linkage, the peptide bond. • Each amino acid has a side chain with distinctive chemical properties. Amino acids may be regarded as the alphabet in which the language of protein structure is written. • The amino acid sequence of a protein constitutes its primary structure, a first level we will introduce within the broader complexities of protein structure. Central Principles in Biochemistry Principle 3 Principle 4 • For study, individual proteins can be separated from the thousands of other proteins present in a cell, based on differences in their chemical and functional properties arising from their distinct amino acid sequences. • Shaped by evolution, amino acid sequences are a key resource for understanding the function of individual proteins and for tracing broader functional and evolutionary relationships. • As proteins are central to biochemistry, the purification of individual proteins for study is a quintessential biochemical endeavor. Amino Acids Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by a specific type of covalent bond. The first to be discovered was asparagine, in 1806. The last of the 20 to be found, threonine, was not identified until 1938. Amino Acids Share Common Structural Features • α carbon and four substituents • α carbon is the chiral center • Tetrahedral • Four substituents: • • • • A carboxyl group An amino group A hydrogen atom An R group (a side chain unique to each amino acid) • Glycine has a second hydrogen atom instead of an R group • Three letter abbreviations and one-letter symbols R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water. The Amino Acid Residues in Proteins are L Stereoisomers • Two possible stereoisomers = enantiomers • Optically active, they rotate the plane of planepolarized light • Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L. • • The amino acid residues in protein molecules are almost all L stereoisomers, with less than 1% being found in the D-configuration. The rare D-amino acid residues generally have a precise structural purpose, and they are introduced to a protein by enzyme-catalyzed reactions that occur after the proteins are synthesized on a ribosome. Amino Acids Can Be Classified by R Group • Five main classes: (based on the properties of their R groups, particularly their polarity, or tendency to interact with water at biological pH (near pH 7.0). • • • • • Nonpolar, aliphatic (7) Aromatic (3) Polar, uncharged (5) Positively charged (3) Negatively charged (2) Amino Acids Can Be Classified by R Group Nonpolar, aliphatic R groups Aromatic R Groups • The hydrophobic effect stabilizes protein structure • R groups absorb UV light at 270–280 nm • Can contribute to the hydrophobic effect Amino Acids Can Be Classified by R Group Polar, Uncharged R Groups • R groups can form hydrogen bonds (more hydrophilic) • Cysteine can form disulfide bonds Amino Acids Can Be Classified by R Group The most hydrophilic R groups are those that are either positively charged or negatively charged. Positively Charged R Groups Negatively Charged R Groups • Have significant positive charge at pH 7.0 • Have a net negative charge at pH 7.0 Histidine, which has an aromatic imidazole group. As the only common amino acid having an ionizable side chain with pKa near neutrality, histidine may be positively charged (protonated form) or uncharged at pH 7.0. His residues facilitate many enzyme-catalyzed reactions by serving as proton donors/acceptors. Uncommon Amino Acids Also Have Important Functions • Modifications of common amino acids: – Modified after protein synthesis (e.G., 4hydroxyproline, found in collagen) – Modified during protein synthesis (e.G., Pyrrolysine, contributes to methane biosynthesis) – Modified transiently to change protein’s function (e.G., Phosphorylation) • Some 300 additional amino acids have been found in cells. • Free metabolites (e.G., Ornithine, intermediate in arginine biosynthesis) Amino Acids Can Act as Acids or Bases • Amino groups, carboxyl groups, and ionizable R groups = weak acids and bases • Zwitterion occurs at neutral pH Nonionic and zwitterionic forms of amino acids Titration of Amino Acids • Acid-base titration involves the gradual addition or removal of protons • —COOH has an acidic pKa (pK1) • —NH3+ has a basic pKa (pK2) • The pH at which the net electric charge is zero is the isoelectric point (pI) Cation ⇌ zwitterion ⇌ anion From the titration curve of glycine we can derive several important pieces of information. (the diprotic form of glycine)  A quantitative measure of the pka of each of the two ionizing groups  Two regions of buffering power  The relationship between its net charge and the ph of the solution.  At ph 5.97, the point of inflection between the two stages in its titration curve, glycine is present predominantly as its dipolar form, fully ionized but with no net electric charge Effect of the Chemical Environment on pKa • α-carboxyl group is more acidic than in carboxylic acids • α-amino group is less basic than in amines Amino Acids as Buffers • Buffers prevent changes in pH close to the pKa • Glycine has two buffer regions: – Centered around the pKa of the αcarboxyl group (pK1 = 2.34) – Centered around the pKa of the α-amino group (pK2 = 9.6) Isoelectric Point, pI • For amino acids without ionizable side chains, the isoelectric point (pI) is: • pH = pI = net charge is zero (amino acid least soluble in water, does not migrate in electric field) • pH > pI = net negative charge • pH < pI = net positive charge Amino Acids Differ in Their Acid-Base Properties • Ionizable side chains: – Have a pKa value – Act as buffers – Influence the pI of the amino acid – Can be titrated (titration curve has 3 ionization steps) Titration of Amino Acids with an Ionizable R Group Finally, in an aqueous environment, only histidine has an R group (pKa = 6.0) providing significant buffering power near the neutral pH usually found in the intracellular and extracellular fluids of most animals and bacteria Titration curves for (a) glutamate and (b) histidine. Peptides and Proteins Biologically occurring polypeptides range in size from small, consisting of two or three linked amino acid residues, to very large, consisting of thousands of residues. Peptides Are Chains of Amino Acids • Peptide bond: • Covalent • Formed through condensation • Broken through hydrolysis • Dipeptide = 2 amino acids, 1 peptide bond • Tripeptide = 3 amino acids, 2 peptide bonds • Oligopeptide = a few amino acids • Polypeptide = many amino acids, molecular weight < 10 kda • Protein = thousands of amino acids, molecular weight > 10 kda length of naturally occurring peptides = 2 to many thousands of amino acid residues *full amino acid names: serylglycyltyrosylalanylleucine Peptide Terminals *three-letter code abbreviations: Ser–Gly–Tyr–Ala–Leu *one-letter code abbreviation: • Numbering (and naming) starts from the amino-terminal residue (N-terminal) N-terminal SGYAL C-terminal Peptides Can Be Distinguished by Their Ionization Behavior • When an amino acid becomes a residue in a peptide, its chemical environment is altered, and the pKa value for an ionizable R group can change somewhat. • Ionizable groups in peptides: • One free α-amino group • One free α-carboxyl group • Some R groups Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which the net charge is zero and they do not move in an electric field. Alanylglutamylglycyllysine. This tetrapeptide has one free αamino group, one free α-carboxyl group, and two ionizable R groups. The groups ionized at pH 7.0 are in red. Peptide Subunits • Multisubunit protein = 2+ polypeptides associated noncovalently • Estimating the Number of Amino Acid Residues • Number of residues = molecular weight/110 • Oligomeric protein = at least 2 identical subunits – identical units = protomers • Average molecular weight of amino acid = ~128 • Amino acid composition is highly variable in proteins • Molecule of water removed to form peptide bond = 18 128 – 18 = 110 *Amino acids are called residues when two or more amino acids bond with each other Some Proteins Contain Chemical Groups Other Than Amino Acids • Conjugated proteins = contain permanently associated chemical components – non–amino acid part = prosthetic group • Lipoproteins contain lipids • Glycoproteins contain sugars • Metalloproteins contain specific metals Some proteins contain more than one prosthetic group. Usually the prosthetic group plays an important role in the protein’s biological function. *A prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins. Working with Proteins To study a protein in detail, the researcher must be able to separate it from other proteins in pure form and must have the techniques to determine its properties. Proteins Can Be Separated and Purified • Separated based on: – Size – Charge – Binding properties – Protein solubility A pure preparation is usually essential before a protein’s properties and activities can be determined. Methods for Purifying Proteins • First step = break open tissue or microbial cells – Crude extract = releases proteins in solution • Second step = fractionation = separate proteins into fractions based on size or charge – “Salting out” = lower solubility of proteins in salt to selectively precipitate proteins (low-speed centrifugation) • Third step = dialysis = use semipermeable membrane to separate proteins from small solutes Column Chromatography … which takes advantage of differences in protein charge, size, binding affinity, and other properties • First step = buffered solution (mobile phase) migrates through porous solid material (solid phase) • Second step = buffered solution containing protein migrates through solid phase • Protein properties affect migration rates Individual proteins migrate faster or more slowly through the column, depending on their properties. Ion-Exchange Chromatography • Separates based on sign and magnitude of the net electric charge • pH and concentration of free salt ions affect protein affinity • The column matrix is a synthetic polymer (resin) containing bound charged groups; • cation exchanger • anion exchangers Size-Exclusion Chromatography • Also called gel filtration chromatography • Separates based on size • Large proteins emerge from the column before small proteins do Affinity Chromatography The beads in the column have a covalently attached chemical group called a ligand — a group or molecule that binds to a macromolecule such as a protein. • Separates based on binding affinity • Eluted by high concentration of salt or ligand High-Performance Liquid Chromatography • Uses high-pressure pumps to move proteins down the column • Greatly improves resolution In most cases, several different methods must be used sequentially to purify a protein completely, each separating proteins on the basis of different properties. As each purification step is completed, the sample size generally becomes smaller, making it feasible to use more sophisticated (and expensive) chromatographic procedures at later stages. Proteins Can Be Separated and Characterized by Electrophoresis • Electrophoresis = visualize and characterize purified proteins • Can be used to estimate: – Number of different proteins in a mixture – Degree of purity – Isoelectric point – Approximate molecular weight This method does not itself contribute to purification, as electrophoresis often adversely affects the structure and thus the function of proteins. • Uses cross-linked polymer polyacrylamide gels • Proteins migrate based on charge-to-mass ratio • Visualization = Coomassie blue dye binds to proteins Sodium Dodecyl Sulfate (SDS) • Sodium dodecyl sulfate (SDS) = a detergent • Binds and partially unfolds proteins • Gives all proteins a similar charge-tomass ratio • Electrophoresis in the presence of SDS separates proteins by molecular weight • Smaller proteins migrate more rapidly • Nearly one molecule of SDS for each amino acid residue Estimating the Molecular Weight of a Protein • Plot of log Mr of marker proteins vs. relative migration during electrophoresis = linear Using Isoelectric Focusing to Determine the pI of a Protein Two-Dimensional Electrophoresis Combining isoelectric focusing and SDS electrophoresisc sequentially. Horizontal separation reflects differences in pI; vertical separation reflects differences in molecular weight. The original protein complement is thus spread in two dimensions. • Permits resolution of complex protein mixtures of proteins • More sensitive than individual methods Unseparated Proteins Are Detected and Quantified Based on Their Functions • Can monitor enzyme purification by assaying specific activity: – Activity = total enzyme units in a solution – Specific activity = number of enzyme units per mg of total protein The Structure of Proteins: Primary Structure Levels of Structure in Proteins The primary structure of a protein determines how it folds up into its unique three-dimensional structure, and this in turn determines the function of the protein. • Four levels: • Primary structure = covalent bonds linking amino acid residues in a polypeptide chain • Secondary structure = recurring structural patterns • Tertiary structure = 3D folding of polypeptide • Quaternary structure = 2+ polypeptide subunits The Function of a Protein Depends on Its Amino Acid Sequence • Amino acid sequence confers 3D structure • 3D structure confers function • Most human proteins = polymorphic = have amino acid sequence variants • Edman degradation = classic method of sequencing amino acids • Traditional protein sequencing techniques: – labeling proteins – breaking proteins into parts Each type of protein has a unique amino acid sequence that confers a particular three dimensional structure. This structure in turn confers a unique function Studying Protein Structure through Labeling • FDNB, dansyl chloride, dabsyl chloride = label amino-terminal α-amino group and εamino group of lysine residues Studying Protein Structure through Breaking Bonds • Oxidation with performic acid or reduction by dithiothreitol= breaks disulfide bonds, denatures proteins Studying Protein Structure Using Proteases • Proteases = catalyze hydrolytic cleavage of peptide bonds Mass Spectrometry Provides Information on Molecular Mass, Amino Acid Sequence, and Entire Proteomes • Mass spectrometry = measure molecular mass with high accuracy – Can sequence short amino acid sequences (20 to 30 amino acid residues) – Can document the entire cellular proteome Small Peptides and Proteins Can Be Chemically Synthesized • The Merrifield method • One end of peptide = attached to resin in column • Protective chemical groups block unwanted reactions Amino Acid Sequences Provide Important Biochemical Information • Amino acid sequence can inform: – 3D structure – Function – Cellular location – Evolution • Consensus sequence = reflects most common amino acid at each position Protein Sequences Help Elucidate the History of Life on Earth • Bioinformatics: – Identifies functional segments in new proteins – Establishes sequence and structural relationships to known proteins • Essential amino acid residues = conserved over evolutionary time • Less important amino acid residues = vary over evolutionary time Transfer of Genes from Organism to Organism • Horizontal gene transfer = transfer of a gene or group of genes from one organism to another – Proteins derived from transferred genes are not good candidates for bacterial evolution studies – For example, rapid spread of antibiotic-resistance genes in bacterial populations Defining Members of Protein Families • Homologs = homologous proteins = members of protein families – Paralogs = homologs in same species – Orthologs = homologs in different species – Identified by comparing protein sequences to a database of protein sequences Constructing Evolutionary Trees • Segregate organisms into classes based on sequence divergence in protein families • Signature sequences = certain protein segments specific to a taxonomic group

Week 3-Amino acids-Peptides-Proteins - Short (1).pptx

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