Chapter 3 AMINO ACIDS, PEPTIDES, AND PROTEINS

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Chapter 3AMINO ACIDS, PEPTIDES, AND PROTEINS
Lehninger Principles of Biochemistry, Fourth
Edition, 2005

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3.1 Amino Acids 3.2 Peptides and Proteins 3.3
Working with Proteins 3.4 The Covalent Structure
of Proteins 3.5 Protein Sequences and Evolution
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Amino Acids share common structural Features

• Proteins are polymers of amino acids, with each
  amino acid residue joined to its neighbor by a
  specific type of covalent bond.
• The a-carbon of AA is a chiral center. Molecules
  with a chiral center are optically active, they
  rotate plane-polarized light.
• The additional carbons in an R group are
  designated b, g, d, e etc.
• Carbon atoms are numbered from one end, giving
  priority to carbons with substitutions.
  containing atoms with the highest atomic numbers.

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D, L System (levorotatory vs. dextrorotatory)

• Enantiomers -nonsuperimposable mirror images of
  each other the two forms represent a class of
  stereoisomers.
• The absolute configurations of simple sugars and
  amino acids are specified by the D, L system.
• RS system is used in the systematic nomenclature
  of organic chemistry and describes more precisely
  the configuration of molecules with more than one
  chiral center.
• 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 exclusively
  L-stereoisomers. D-Amino acid residues have been
  found only in a few, generally small peptides,
  including some peptides of bacterial cell walls
  and certain peptide antibiotics.

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The R groups in this class of amino acids are
nonpolar and hydrophobic.
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Nonpolar, Alipatic R Groups

• Glycine has the simplest structure, its very
  small side chain makes no real contribution to
  hydrophobic interactions
• Methionine, one of two sulfur containing amino
  acids. has a nonpolar thioether group. First AA
  residue in translation of proteins
• Alanine, Valine, Leucine, and Isoleucine could
  contribute to hydrophobic interaction.
• The secondary amino (imino) group of Pro is held
  in a rigid conformation that reduces the
  structural flexibility of polypeptide regions
  containing proline.

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Absorbance of UV by Aromatic Amino acids

• All are relatively nonpolar (hydrophobic).
• -OH group of throsine can form hydrogen bonds and
  are important functional group. Can be
  phosphorylated as well.
• All can absorb UV light (280 nm), Tyrosine and
  Tryptophan are stronger than phenylalanine. Use
  for protein quantification.

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Polar, Uncharged R Groups

• The R groups of these amino acids are more
  soluble in water, or more hydrophilic, than those
  of the nonpolar amino acids, because they contain
  functional groups that form hydrogen bonds with
  water.
• Serine and Threonine has OH, which contribute to
  polarity, and could be phosophorelated.
• Asparagine and Glutamine are the amides of
  Aspartate, and Glutamate, and are easily
  hydrolyzed by acid or base.

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Reversible Formation of Disulfide Bond

• Cysteine is readily oxidized to form a covalently
  linked dimeric AA called cystine (disulfide
  bond).
• The disulfide-linked residues are strongly
  hydrophobic (nonpolar). Disulfide bonds play a
  special role in the structures of many proteins
  by forming covalent links between parts of a
  protein molecule or between two different
  polypeptide chains.

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Positively Charged (Basic) R Groups
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Positively Charged (Basic) R Groups

• Lysine has a second primary amino group at e
  position. Its R group has significant positive at
  pH7.
• Arginine has a positively charged guanidino group
• Histidine a imidazole group, and is the only
  standard amino acid having an ionizable side
  chain with a pKa near neutrality. It serves as a
  proton donor/ acceptor in a enzyme-catalyzed
  reaction

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Negatively Charged (Acidic) R Groups

• Two amino acids having R groups with a net
  negative charge at pH7 are asparate and
  glutamate, each of which has a second carboxyl
  group

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Classify the amino acids by polarity
Juang RH (2003) Biochemistry
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Uncommon Amino Acids - I
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Nonstandard Amino Acids

• Some 300 additional amino acids have been found
  in cells.
• Are created by modification of standard residues
  already incorporated into a peptide.
• 4-hydroxyproline, a derivative of proline, is
  found in plant cell wall protein and collagen
  5-hydroxylysine, derived from lysine, are found
  in collagen.
• 6-N Methyllysine is a constituent of myosin.
• g-Carboxyglutamate, found in the blood-clotting
  protein prothrombin and Ca2 binding protein.
• Desmosine, derivative of four Lys residues, which
  is found in the elastin.
• Selenocysteine is introduced during protein
  synthesis, and contains Selenium rather than
  sulfur of cysteine, derived from serine.

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Uncommon Amino Acids - II

• Ornithine and citrulline are not constituents of
  proteins.
• They are key intermediates (metabolites) in the
  biosynthesis of arginine and in the urea cycle.

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Amino acids Can Act as Acids and Bases

• Zwitterion (hybrid ion) dipolar ion, can act as
  either an acid (proton donor or a base (proton
  acceptor) - Amphoteric mater ampholyte
  (amphoteric electrolytes)

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Amino Acids Have Characteristic Titration Curves

• The pKa is a measure of the tendency of a group
  to give up a proton, with the tendency decreasing
  tenfold as the pKa increases by on unit.

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Titration Curves Predict the Electric Charge of
Amino Acids

• Isoelectric point (isoelectric pH) pI, The
  characteristic pH at which the net electric
  charge is zero (eg. glycine).

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Titration Curves of Glutamate
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Titration Curves of Histidine
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Effect of the chemical environment on pKa
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Effect of the chemical environment on pKa

• The perturbed pKa of glycine is caused by
  repulsion between the departing proton and the
  nearby positively charged amino group. The
  opposite charges on the resulting zwitterion are
  stabilizing, nudging the equilibrium farther to
  the right.
• The electronegative oxygen atoms in the carboxyl
  groups, which tend to pull electrons toward them,
  increasing the amino group to give up a proton.

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3.2 Peptides and Proteins
Formation of a Peptide Bond by Condensation
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The pentapeptide serylglycyltyrosylalanylleucine
(SerGlyTyrAlaLeu)
C terminal
N terminal

• A few amino acids are joined - an oligopeptide.
• Many amino acids are joined, - a polypeptide.
• Protein and polypeptide are sometimes used
  interchangeably, molecules referred to as
  polypeptides generally have molecular weights
  below 10,000 (D), and those called proteins have
  higher molecular weights.

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Peptides Can Be Distinguished by Their Ionization
Behavior (Alanyl-glutamyl-glycyl-lysine)

• The acid-base behavior of a peptide can be
  predicted from its free -amino and -carboxyl
  groups as well as the nature and number of its
  ionizable R groups.
• Peptides have characteristic titration curves and
  a characteristic isoelectric pH (pI) at which
  they do not move in an electric field.

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Biologically Active Peptides and Polypeptides
Occur in a Vast Range of Sizes

• Titin, a constituent of vertebrate muscle, which
  has 27,000 AAs, and M.W.3,000,000.
• Single peptide chain Vs. multisubunit protein
  two or more polypeptide associated noncovalently.
  
• The individual polypeptide chains in a
  multisubunit
• protein may be identical or different. If at
  least two
• are identical the protein is said to be
  oligomeric, and the identical units (consisting
  of one or more polypeptide chains) are referred
  to as protomers.
• Ex. Hemoglobin- has four polypeptide subunits
  two identical a chains and two identical b
  chains, all four held together by noncovalent
  interactions. Each subunit is paired in an
  identical way with a subunit within the
  structure of this multisubunit protein, so that
  hemoglobin can be considered either a tetramer of
  four polypeptide subunits or a dimer of ab
  protomers.
• The average M.W. of AA 110 (128-18)

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Levels of Structure in Protein

• Primary A description of all covalent bonds. The
  sequence of AA residues
• Secondary particularly stable arrangements of AA
  giving rise to recurring structural patterns.
• Tertiary All aspects of the 3D folding of a
  polypeptide.
• Quaternary The spatial arrangement of
  multisubunits protein

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3.3 Working with Proteins
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Separation and Purification of Proteins

• Including size, charge, and binding properties.
• Crude extract breaking cells, by osmosis lysis
  or homogenization.
• Fractionation separate proteins into different
  fraction based on size of charge.
• Salting out The solubility of proteins is
  lowered at high salt concentration. Ammonium
  sulfate ((NH4)2SO4).
• Dialysis is a procedure to separate proteins from
  solvents

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A Purification Table for a Hypothetical Enzyme

• 1. Crude cellular extract
• 2. Precipitation with ammonium sulfate
• 3. Ion-exchange chromatography
• 4. Size-exclusion chromatography
• 5. Affinity chromatography
• Fraction volume (ml)
• Total protein (mg)
• Activity (units)
• Specific activity (units/mg)

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Protein Purification Column Chromatography

• The expansion of the protein band in the mobile
  phase is caused by separation of proteins with
  different properties and by diffusion spreading.
  As the length of the column increases, the
  resolution of two types of protein improves.
• Rate is decreased and resolution can decline
  because of the diffusion spreading.
• HPLC, or high-performance liquid chromatography.
  uses high-pressure pumps that
• speed the movement of the protein molecules down
  the column, as well as higher-quality
  chromatographic materials that can withstand the
  crushing force of the pressurized flow. By
  reducing the transit time on the column, - limit
  diffusional spreading of protein bands and thus
  greatly improve resolution.

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Ion-exchange Chromatography (net electric charges)

• The column matrix is a synthetic polymer
  containing bound charged groups those with bound
  anionic groups (negatively charged) are called
  cation exchangers,
• bound cationic groups (positively charged) are
  called anion exchangers.
• Is effected by pH and salt concentration.

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Size-Exclusion Chromatography (size)

• Also called gel filtration chromatography
• The column matrix is a cross-linked polymer with
  pores of selected size.
• Larger protein migrate faster than smaller ones
  because they are too large to enter the pores

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Affinity Chromatography (binding specificity)

• separates proteins by binding specificities.
• The proteins retained on the column are those
  that bind specifically to a ligand cross-linked
  to the beads.
• After proteins that do not bind to the ligand are
  washed through the column, the bound protein of
  particular interest is eluted by a solution
  containing free ligand.

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High Performance Liquid Chromatography (HPLC)
use of high pressure to push a mobile phase
solution through a column of stationary phase
allowing separation of complex mixtures with high
resolution.
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Normal vs. Reversed Phase Chromatography
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Electrophoresis

• Separation of proteins is based on the migration
  of charged protein in an electric field
• The migration of a protein in a gel during
  electrophoresis is a function of its size and
  shape.
• m V / E Z / f
• m The electrophoretic mobility
• V velocity
• E electrical potential
• Z net charge
• f frictional coefficient (shope)

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SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis

• SDS binds to most proteins probably by
  hydrophobic interaction. One SDS for every two
  AAs, Thus, each protein has a similar
  charge-to-mass ratio.
• Stains protein Coomassie blue, Silver, and Sypro
  Ruby
• Western blot

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Estimating the Molecular Weight of a Protein
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Isoelectric Focusing

• pI of a protein net charge0
• A pH gradient is established by allowing a
  mixture of organic acids and bases (ampholytes).
  Protein migrates until it reaches the pH that
  matches its pI

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Two-Dimensional Electrophoresis

• Separates proteins of identical MW that differ in
  pI or proteins with similar pI but different MW.

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Two-dimensional Gel Electrophoresis
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Staining of Polyacrylamide Gels
Silver staining
Coomassie blue staining
Sypro Ruby staining
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Activity vs. Specific Activity

• Unit amount of enzyme causing transformation of
  1 m mole of substrate per min. at 25 oC under
  optimal conditions
• Activity Total units of enzyme (U).
• Specific activity Activity of total protein
  (U/mg)

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3.4 The Covalent Structure of Proteins
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The Function of a Protein Depends on Its Amino
acid Sequence

• Proteins with different function have different
  AA sequence
• Altering primary structure changes the function
  of proteins
• Similar proteins from different species have
  similar AA sequences.
• Function of a protein depends on its structure
  Structure depends on sequence.
• Polymorphic (polymorphism) AA sequence
  variation- An estimated 20 to 30 of the
  proteins are polymorphic in population.
• Specific region (Domain)

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Insulins difference by species
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Protein Sequencing (I) Breaking Disulfide Bonds
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Protein SequencingCleaving the Polypeptide Chain
protease
Tyrosine
Chymotrypsin
Pepsin
CNBr
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Protein sequencing (3) Sangers Method vs.
Edman degradation
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Cleaving proteins and sequencing and ordering the
peptide fragments
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Investigating Proteins with Mass
SpectrometryElectrospray ionization mass
spectrometry (ESI)
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Matrix-assisted laser desorption/ionization mass
spectrometry (MALDI MS)
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Mass Spectrometric Identification of Proteins -
Mapping
Peptide mass fingerprinting (PMF) or peptide
mapping
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Obtaining protein sequence information with
tandem MS
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How to 0brain a peptide or protein?

• purification from tissue, a task often made
  difficult by the vanishingly low concentrations
  of some peptides
• genetic engineering
• Direct chemical synthesis.

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Chemical Synthesis of Peptide (R. Bruce
Merrifield)
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Error Rate is Increased as The Polypeptide Chain
Gets Longer
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Protein Sequences Can Elucidate the History of
Life on Earth

• For a given protein, the amino acid residues
  essential for the activity of the protein are
  conserved over evolutionary time. The residues
  that are less important to function may vary over
  time
• If two organisms are closely related, the
  sequences of their genes and proteins should be
  similar. The sequences increasingly diverge as
  the evolutionary distance between two organisms
  increases.
• The members of protein families are called
  homologous proteins, or homologs - Paralogs
  (same species) vs. Orthologs (different species)
• the rare transfer of a gene or group of genes
  from one organism to another, a process called
  lateral gene transfer.

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Protein Homology among Species

• Invariant (conserved) residues (same residue) vs.
  variable residues.
• Conservative substitutions Substitutions with
  similar amino acid residue (i.e. Arg to Lys).
• The number of residues that differ in homologous
  protein from any two species is in proportion to
  the phylogenetic (evolutionary) difference
  between those species.

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Aligning protein sequences with the use of gaps

• Within this sequence alignment, a positive score
  is assigned for each position where the amino
  acid residues in the two sequences are identical.
• In some case, two sequence segments are connected
  by less related sequences of different lengths -
  cannot be aligned at the same time introduce
  gap (penalties negative score)
• When amino acid substitutions are found within a
  protein family, many of the differences may be
  conservative - that is, an amino acid residue is
  replaced by a residue having similar chemical
  properties. Ex. Glu Asp Leu Ala.

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Blosum (blocks substitution matrix) table
Blosum62

• The identical residues were given scores based on
  how often they were replaced, such that amino
  acids with unique chemical properties (such as
  Cys and Trp) received higher scores than those
  more conservatively replaced (such as Asp and
  Glu).

• Higher scores were given to nonidentical residues
  that occurred frequently than to those that
  appeared rarely.

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Signature sequences in the EF-1/EF-Tu protein
family

• Certain segments of a protein sequence may be
  found in the organisms of one taxonomic group but
  not in other groups these segments can be used
  as signature sequences for the group in which
  they are found.

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Evolutionary tree derived from amino acid
sequence comparisons
external nodes - Extant species
internal nodes Extinct ancestor species
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Electrophoresis
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PAGE ?????

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• ???? (acrylamide),H2CCH-CO-NH2?
• Acrylamide ???? Bis ??????,????,????????
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• Bis N,N'-methylene-bis(acrylamide)
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• TEMED (tetramethylethylenediamine) ??????????

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