Chapter 3: Amino Acids, Peptides, and Proteins Dr. Rajabi

Outline (part I) Sections 3.1 and 3.2 Amino Acids Chemical structure Acid-base properties Stereochemistry Non-standard amino acids Formation of Peptide Bonds

Amino Acids The building blocks of proteins Also used as single molecules in biochemical pathways 20 standard amino acids (a-amino acids)( 21 amino acid=Selenocysteine) Two functional groups: carboxylic acid group amino group on the alpha () carbon Have different side groups (R) Properties dictate behavior of AAs R side chain | H2N— C —COOH | H

Zwitterions Both the –NH2 and the –COOH groups in an amino acid undergo ionization in water. At physiological pH (7.4), a zwitterion forms Both + and – charges Overall neutral Amphoteric Amino group is protonated Carboxyl group is deprotonated Soluble in polar solvents due to ionic character Structure of R also influence solubility

Classification of Amino Acids Classify by structure of R Nonpolar Polar Aromatic Acidic Basic

Nonpolar Amino Acids Hydrophobic, neutral, aliphatic

Polar Amino Acids Hydrophilic, neutral, typically H-bond

Disulfide Bonds Formed from oxidation of cysteine residues

Aromatic Amino Acids Bulky, neutral, polarity depend on R

Acidic and Basic Amino Acids R group = carboxylic acid Donates H+ Negatively charged Basic R group = amine Accepts H+ Positively charged His ionizes at pH 6.0

Acid-base Properties Remember H3PO4 (multiple pKa’s) AAs also have multiple pKa’s due to multiple ionizable groups pK1 ~ 2.2 (protonated below 2.2) pK2 ~ 9.4 (NH3+ below 9.4) pKR (when applicable)

Table 3-1 Note 3-letter and 1-letter abbreviations Amino acid organization chart

pH and Ionization Consider glycine: Note that the uncharged species never forms

Titration of Glycine pK1 pK2 First equivalence point Animation [cation] = [zwitterion] pK2 [zwitterion] = [anion] First equivalence point Zwitterion Molecule has no net charge pH = pI (Isoelectric point) pI = average of pKa’s = ½ (pK1 + pK2) pIglycine = ½ (2.34 + 9.60) = 5.97 Animation

pI of Lysine For AAs with 3 pKa’s, pI = average of two relevant pKa values Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53): Which species is the isoelectric form? So, pI = ½ (pK2 + pKR) = ½ (8.95 + 10.53) = 9.74 Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)

Learning Check Would the following ions of serine exist at a pH above, below, or at pI?

 Histidine A good buffer at ~ pH 6. pI =

Stereochemistry of AAs All amino acids (except glycine) are optically active Fischer projections:

D and L Configurations d = dextrorotatory l = levorotatory D, L = relative to glyceraldehyde

Importance of Stereochemistry All AA’s found in proteins are L geometry S enantiomer for all except cysteine D-AA’s are found in bacteria Geometry of proteins affects reactivity (e.g binding of substrates in enzymes) Thalidomide

Non-standard Amino Acids AA derivatives Modification of AA after protein synthesized Terminal residues or R groups Addition of small alkyl group, hydroxyl, etc. D-AAs Bacteria

CHEM 2412 Review Carboxylic acid + amine = ? Structure of amino acid

The Peptide Bond Chain of amino acids = peptide or protein Amino acid residues connected by peptide bonds Residue = AA – H2O

The Peptide Bond Non-basic and non-acidic in pH 2-12 range due to delocalization of N lone pair Amide linkage is planar, NH and CO are anti Rigid restricted rotation

Polypeptides Linear polymers (no branches) AA monomers linked head to tail Terminal residues: Free amino group (N-terminus) Draw on left Free carboxylate group (C-terminus) Draw on right pKa values of AAs in polypeptides differ slightly from pKa values of free AAs

Learning Check Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.

Learning Check Draw the structural formula of each of the following peptides. A. Methionylaspartic acid B. Alanyltryptophan C. Methionylglutaminyllysine D. Histidylglycylglutamylalanine

Outline (part II) Sections 3.3 and 3.4 Separation and purification Protein sequencing Analysis of primary structure

Protein structure: Primary structure: There are four levels of protein structure (primary, secondary, tertiary and quaternary) Primary structure:   The primary structure of a protein is its unique sequence of amino acids. Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids. The precise primary structure of a protein is determined by inherited genetic information. At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus).

2- Secondary structure: Results from hydrogen bond formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure: α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).

I →I+4

Hydrogen bonding in α-helix: In the α-helix CO of the one amino acid residue forms H-bond with NH of the forth one. Supersecondary structure or Motifs : occurs by combining secondary structure. The combination may be: α-helix- turn- α-helix- turn…..etc Or: β-sheet -turn- β-sheet-turn………etc Or: α-helix- turn- β-sheet-turn- α-helix Turn (or bend): is short segment of polypeptides (3-4 amino acids) that connects successive secondary structures. e.g. β-turn: is small polypeptide that connects successive strands of β-sheets.

Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure.

Quaternary structure: results from the aggregation (combination) of two or more polypeptide subunits held together by non-covalent interaction like H-bonds, ionic or hydrophobic interactions. Examples on protein having quaternary structure: Collagen is a fibrous protein of three polypeptides (trimeric) that are supercoiled like a rope. This provides the structural strength for their role in connective tissue. Hemoglobin is a globular protein with four polypeptide chains (tetrameric) Insulin : two polypeptide chains (dimeric)

Summary of Protein Structures Copyright © 2007 by Pearson Education, Inc Publishing as Benjamin Cummings

Protein size In general, proteins contain > 40 residues Minimum needed to fold into tertiary structure Usually 100-1000 residues Percent of each AA varies Proteins separated based on differences in size and composition Proteins must be pure to analyze, determine structure/function

Factors to control pH Presence of enzymes Temperature Thiol groups Keep pH stable to avoid denaturation or chemical degradation Presence of enzymes May affect structure (e.g. proteases/peptidase) Temperature Control denaturation (0-4°C) Control activity of enzymes Thiol groups Reactive Add protecting group to prevent formation of new disulfide bonds Exposure to air, water Denature or oxidize Store under N2 or Ar Keep concentration high

General Separation Procedure Detect/quantitate protein (assay) Determine a source (tissue) Extract protein Suspend cell source in buffer Homogenize Break into fine pieces Cells disrupted Soluble contents mix with buffer Centrifuge to separate soluble and insoluble Separate protein of interest Based on solubility, size, charge, or binding ability

Solubility Selectively precipitate protein Manipulate Concentration of salts Solvent pH Temperature

Concentration of salts Adding small amount of salt increases [Protein] Salt shields proteins from each other, less precipitation from aggregation Salting-in Salting out Continue to increase [salt] decreases [protein] Different proteins salt out at different [salt]

Other Solubility Methods Solvent Similar theory to salting-out Add organic solvent (acetone, ethanol) to interact with water Decrease solvating power pH Proteins are least soluble at pI Isoelectric precipitation Temperature Solubility is temperature dependent

Chromatography Mobile phase Stationary phase Mixture dissolved in liquid or solid Stationary phase Porous solid matrix Components of mixture pass through the column at different rates based on properties

Types of Chromatography Paper Stationary phase = filter paper Same theory as thin layer chromatography (TLC) Components separate based on polarity High-performance liquid (HPLC) Stationary phase = small uniform particles, large surface area Adapt to separate based on polarity, size, etc. Hydrophobic Interaction Hydrophobic groups on matrix Attract hydrophobic portions of protein

Types of Chromatography Ion-exchange Stationary phase = chemically modified to include charged groups Separate based on net charge of proteins Anion exchangers Cation groups (protonated amines) bind anions Cation exchangers Anion groups (carboxylates) bind cations

Types of Chromatography Gel-filtration Size/molecular exclusion chromatography Stationary phase = gels with pores of particular size Molecules separate based on size Small molecules caught in pores Large molecules pass through

Types of Chromatography Affinity Matrix chemically altered to include a molecule designed to bind a particular protein Other proteins pass through

UV-Vis Spectroscopy Absorbance used to monitor protein concentrations of each fraction l = 280 nm Absorbance of aromatic side groups

Electrophoresis Migration of ions in an electric field Electrophoretic mobility (rate of movement) function of charge, size, voltage, pH The positively charged proteins move towards the negative electrode (cathode) The negatively charged proteins move toward the positive electrode (anode) A protein at its pI (neutral) will not migrate in either direction Variety of supports (gel, paper, starch, solutions)

Protein Sequencing Determination of primary structure Need to know to determine 3D structure Gives insight into protein function Approach: Denature protein Break protein into small segments Determine sequences of segments Animation

End group analysis Identify number of terminal AAs Number of chains/subunits Identify specific AA Dansyl chloride/dabsyl chloride Sanger method (FDNB) Edman degradation (PITC) Bovine insulin

Dansyl chloride Reacts with primary amines N-terminus Yields dansylated polypeptides Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence) Useful method when protein fragmented into shorter polypeptides

Dabsyl chloride and FDNB Same result as dansyl chloride Dabsyl chloride 1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger method

Edman degradation Phenylisothiocyanate (PITC) Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC) Treat with TFAA (solvent/catalyst) to cleave N-terminal residue Does not hydrolyze other AAs Treatment with dilute acid makes more stable organic compound Identify using NMR, HPLC, etc. Sequenator (entire process for proteins < 100 residues)

Fragmenting Proteins Formation of smaller segments to assist with sequencing Process: Cleave protein into specific fragments Chemically or enzymatically Break disulfide bonds Purify fragments Sequence fragments Determine order of fragments and disulfide bonds

Cleaving Disulfide Bonds Oxidize with performic acid Cys residues form cysteic acid Acid can oxidize other residues, so not ideal

Cleaving Disulfide Bonds Reduce by mercaptans (-SH) 2-Mercaptoethanol HSCH2CH2OH Dithiothreitol (DTT) HSCH2CH(OH)CH(OH)CH2SH Reform cysteine residues Oxidize thiol groups with iodoacetete (ICH2CO2-) to prevent reformation of disulfide bonds

Hydrolysis Cleaves all peptide bonds Achieved by After cleavage: Enzyme Acid Base After cleavage: Identify using chromatography Quantify using absorbance or fluorescence Disadvantages Doesn’t give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results

Enzymatic and Chemical Cleavage Enzymes used to break protein into smaller peptides Endopeptidases Catalyze hydrolysis of internal peptide bonds Chemical Chemical reagents used to break up polypeptides Cyanogen bromide (BrCN)

An example

Another example A protein is cleaved with cyanogen bromide to yield the following sequences: Arg-Ala-Tyr-Gly-Asn Leu-Phe-Met Asp-Met The same protein is cleaved with chymotrypsin to yield the following sequences: Met-Arg-Ala-Tyr Asp-Met-Leu-Phe Gly-Asn What is the sequence of the protein?

Suggested Problems, Chapter 3 1-5, 7, 10-13, 15, 18

2- Secondary structure: Results from hydrogen bond formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure: α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).