CH 5 The Primary Level of Protein Structure

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CH 5 The Primary Level of Protein Structure

• HW 2, 3, 4, 6, 7

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Amino Acids and Peptides

• Bioimportance
• Monomer units for proteins
• Participate in cellular functions as nerve
  transmissions
• Biosynthesis of porphyrins, purines, pyrimidines,
  and urea

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• While human proteins only contain L-amino acids,
  other organisms contain both D and L
• 10 of the 20 amino acids commonly used are
  essential nutrients, which mean they must be in
  our diet, we can not synthesize them
• There are over 300 naturally occurring amino
  acids, but only 20 are used to make proteins.

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• These 20 are listed in table 5.3 page 129,
  according to side chain properties
• Thursday, we will have a quiz on the names, 1
  letter abbreviation, and 3 letter abbreviation
• Next Tuesday, we will have a quiz on the name,
  structure, and pKas
• While we only use 20, some of these can be
  altered in a peptide by adding or removing a
  functional group to increase diversity.

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Chirality

• All amino acids, except Gycine, have an alpha
  carbon that is chiral.
• This is the source of all chirality in organisms
• The absolute configuration of all the alpha
  carbons are L

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Amino Acids properties

• Amino Acids may have a positive, negative or zero
  net charge.
• Most amino acids are in the zwitterionic state at
  physiologic pH
• An amino acid can not exist as COOH/NH2 because
  any pH low enough to protonate the COO- group
  would as protonate the NH2

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• Some amino acids, histidine and arginine, are
  resonance hybrids, but they still only have a 1
  charge.
• By altering the net charge via pH, we can create
  separation processes for amino acids, peptides,
  and proteins
• The isoelectric species is the form of a molecule
  that has an equal number of positive and negative
  charges, thus it is neutral.

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• The isoelectric pH, also called pI, is the pH
  midway between the pKa values on either side of
  the isoelectric species
• Examples

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• This pI guides selection of separation conditions
• The pKa of side chains varies slightly
• Nonpolar effects-
• Thus the pKa in peptides and proteins will depend
  on unique local environments
• These changes in charge affect physical
  properties of amino acids, peptides, and
  proteins.
• Functional groups of the side chains typically
  determine chemical properties

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• When amino acids are in a protein or peptide
  chain, they are called residues
• Peptides are usually written with the free alpha
  amino group to the left and the free alpha
  carboxyl group to the right
• The backbone will start with N, then the alpha
  carbon, then the carbonyl carbon, then repeat.
• The side chains are bonded to the alpha carbon
• Lines are used with 3-letter abbreviations,
  omitted with 1-letter abbreviations

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• For some peptides, non-common amino acids may be
  used or non-peptide bonds my be used
• The peptide bond is not charged, however you
  still have a at the N-terminal, - at the
  C-terminal, and any charges on side chains
• Peptides are therefore classified as
  Polyelectrolytes

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Structure Feature of Peptide Bond

• See figure 5.12 page 138
• The peptide bond has some double bond character
  and does not have rotation about the C-N peptide
  bond
• The carbonyl O and C, the N,and the H on the N,
  all lie in the same plane.
• Rotation only occurs between the alpha carbon and
  N, and alpha carbon and COO-

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Determination of Primary Structure

• We know proteins are very important
• An important goal of molecular medicine is the
  identification of proteins who presence, absence,
  or deficiency is associated with specific
  physiologic states or disease
• The primary structure of proteins, which is the
  sequence of amino acids, provides both a
  molecular finger print for its identification and
  information that can be used to identify and
  clone the gene or genes that encode it.

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• In order to determine the amino acid sequence, a
  protein or peptide must be highly purified
• Because of the 1000s of different proteins in
  each cell, this is very difficult to do.
• It usually requires many successive purification
  techniques

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Purification

• The classic approach exploits differences in
• Relative solubility of proteins as a function of
  pH
• Polarity
• Salt concentration
• Chromatographic separtions

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Chromatographic Separations

• Mobile phase vs stationary phase
• Paper chromatography
• TLC
• Column
• Types of stationary phase
• Size exclusion
• Absorption
• Ion exchange

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Other types of Chromatography

• pH based chromatography
• Hydrophobic Interaction chromatography

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Affinity Chromatography

• Exploits high selectivity of binding proteins
• This is what we did in lab
• All the chromatography mentioned so far, is
  typically done slowly with low pressure
• The stationary phases involved are somewhat
  spongy and their compressibility limit flow
  rates

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HPLC

• High Pressure Liquid Chromatography uses
  incompressible silica or alumina as stationary
  phase which allows much higher flow rates and
  pressures
• This also helps limit diffusion thus enhances the
  resolution
• This method is very effective on complex mixtures
  of lipids or peptides with very similar properties

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HPLC

• The stationary phase is typically hydrophobic and
  water miscible organic solvents such as
  acetonitrile or methanol are used as mobile
  phases

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SDS-PAGE

• SDS- sodium dodecyl sulfate (anion detergent)
• PAGE- poly acrylamide gel electrophoresis
• Electrophoresis separates biomolecules based on
  their ability to move through a gel matrix due to
  an applied electric field
• SDS denatures and binds to proteins at a known
  ratio of 1 SDS for every 2 peptide bonds

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• 2-mercaptoethanol or dithiothreitol is used to
  break disulfide linkages
• The charge on SDS,-1, overcomes and negates
  charges on side chains
• This leads to a constant charge to mass ratio
  which means the peptides are separated purely by
  the resistance the matrix provides
• Larger peptides have more resistance, therefore
  move slower
• The gel is then stained, usually with Coomassie
  Blue, to visualize the movement

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IEF

• IEF- Isoelectric Focusing
• Ionic buffers called ampholytes and applied
  electric field are used to generate a pH gradient
  with in a matrix
• The peptide/protein then migrate through the
  matrix to an area where the pHpI, so there is no
  net charge on the peptide
• IEF can be used in conjunction with SDS-PAGE to
  perform a 2-D analysis, separating peptides first
  by pI, then by size

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Sequencing Peptides

• Sanger was the first to determine the sequence of
  a polypeptide
• Mature insulin consist of 2 chains, the A chain
  has 21 residues, the B chain has 30 residues
• The chains are held together by disulfide
  linkages
• Sanger first broke the linkages to separate the
  chains

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• He then broke the chains into smaller pieces
  using trypsin, chymotrypsin, and pepsin
• These reagents cleave peptide bonds at know
  locations
• These smaller fragments where the separated and
  hydrolyzed to form even smaller peptide chains
• Each was reacted with 1-fluoro-2,4-dinitrobenzene,
  called Sangers reagent, which derivatizes the
  exposed alpha amino group

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• The amino acid content of the peptide was then
  determined.
• Working backwards, he was able to determine the
  complete sequence of insulin and win the Nobel
  Prize in 1958

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Peptide Cleaving Agents

• Reagent Bond Cleaved
• CNBr Met-X
• Trypsin Lys-X and Arg-X
• Chymotrypsin Hydrophobic AA-X
• Endoproteinase Lys-C Lys-X
• Endoproteinase Arg-C Arg-X
• Endoproteinase Asp-N X-Asp

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• Reagent Bond Cleaved
• V8 protease Glu-X particularly where
• X is hydrophobic
• Hydroxylamine Asn-Gly
• o-Iodosobenzene Trp-X
• Mild Acid Asp-Pro

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Edmans Reagent

• Pehr Edman introduced phenylisothiocyanate,
  called Edmans reagent, to selectivity label the
  amino-terminal residue of a peptide
• Unlike Sangers, Edmans derivative can be
  removed under mild conditions with out disrupting
  the rest of the peptide
• After removal, a new amino terminal is produced
  and the process is repeated
• This allows for the direct sequencing of a peptide

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• However, due to the efficiency of the reaction,
  this process is limited to peptides no larger
  than 20-30 residues
• Process

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• Because of the limitations to Edmans process and
  since most polypeptides contain several hundred
  residues, most polypeptides must be broken into
  smaller chains which are then identified.
• Sample problem on hand out.