Protiens and peptids

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Peptides and Proteins

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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abstract

• Overview of amino acids, peptides and the peptide
  bond
• Discuss the levels of protein structure
• Describe techniques used for analysis of proteins

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Planar nature of the peptide bond. The partial
double bond characteristic prevents free rotation
around the C-N bond keeping it in the same plane
with the attached O and H atoms. These planar
bonds can pivot around the shared Ca atom
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Levels of Protein Structure
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Protein Structure Levels

• PRIMARY the linear sequence of amino acids
  linked together by peptide bonds
• SECONDARY regions within polypeptide chains with
  regular, recurring, localized structure
  stabilized by H-bonding between constituent amino
  acid residues

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Protein Structure Levels (cont)

• TERTIARY the overall three-dimensional
  conformation of a protein
• QUATERNARY the three-dimensional conformation of
  a protein composed of multiple polypeptide
  subunits
• THE PRIMARY AMINO ACID SEQUENCE IS THE ULTIMATE
  DETERMINANT OF FINAL PROTEIN STRUCTURE

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Disulfide bonds
Form between two intra- or interchain
cysteine residues, product called cystine -
Stabilizes/creates protein conformation -
Prevalent in extracellular/ secreted proteins
Ex INSULIN
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Stabilizing Forces
1. Electrostatic/ionic 3. Hydrophobic
interactions 2. Hydrogen bonds 4. Disulfide bonds
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(No Transcript)
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2o Structure a-helix each oxygen of a carbonyl
group of a peptide bond forms a H-bond with the
hydrogen atom attached to a nitrogen in a peptide
bond 4 amino acids further along the chain very
stable structurally prolines will disrupt helix
formation
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End-on view of a-helix
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b-sheet
In this secondary structure, each amino acid
residue is rotated 180o relative to its adjacent
residue. Occur most commonly in anti-parallel
directions, but can also be found in parallel.
H-bonds between adjacent chains aid in
stabilizing the conformation.
Anti-Parallel
Parallel
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Super-secondary structure examples
b-bend
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Super-secondary structures commonly found in some
DNA-binding proteins
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Domains, examples
b-Barrel
Bundle
Saddle
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Ex Tertiary Structure
Ex Quaternary Structure
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Myoglobin b-subunit Hemoglobin
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Structure of Myoglobin and Hemoglobin

• The amino acid sequences of myoglobin and
  hemoglobin are similar (or, highly conserved) but
  not identical
• Their polypeptide chains fold in a similar manner
• Myoglobin is found in muscles as a monomeric
  protein hemoglobins are found in mature
  erythrocytes as multi-subunit tetrameric
  proteins. Both are localized to the cytosol

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Sequence Comparison Examples
(Surface helix)
Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb
(human) Hbg (human) Hbd (human)
(Internal helix)
Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb
(human) Hbg (human) Hbd (human)
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Myoglobin Properties

• At the tertiary level, surface residues prevent
  one myoglobin from binding complementarily with
  another myoglobin thus it only exists as a
  monomer.
• Each monomer contains a heme prosthetic group a
  protoporphryin IX derivative with a bound Fe2
  atom.
• Can only bind one oxygen (O2) per monomer
• The normal physiological O2 at the muscle is
  high enough to saturate O2 binding of myoglobin.

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Heme Structure
Protein-Heme Complex with bound oxygen
Heme-Fe2
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Hemoglobin Properties

• At the tertiary level, the surface residues of
  the a and b subunits form complementary sites
  that promote tetramer formation (a2b2), the
  normal physiological form of hemoglobin.
• Contains 4 heme groups, so up to 4 O2 can be
  bound
• Its physiological role is as a carrier/transporter
  of oxygen from the lungs to the rest of the
  body, therefore its oxygen binding affinity is
  much lower than that of myoglobin.
• If the Fe2 becomes oxidized to Fe3 by chemicals
  or oxidants, oxygen can no longer bind, called
  Methemoglobin

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Biochemical Methods to Analyze Proteins

• Electrophoresis
• Chromatography Gel filtration, ion exchange,
  affinity
• Mass Spectrometry, X-ray Crystallography, NMR
• You will not be tested on the sections in your
  textbook describing amino acid separations (Ch
  4), peptide/protein sequencing and synthesis (Ch
  5), and X-ray crystallography/NMR (Ch 6)

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Protein Separation by SDS-Polyacrylamide Gel
Electrophoresis
Presence of SDS, a detergent, denatures and
linearizes a protein (Na and sulfate bind to
charged amino acids, the hydrocarbon chain
interacts with hydrophobic residues). An applied
electric field leads to separation of proteins
based on size through a defined gel pore matrix.
For electrophoresis in the absence of SDS,
separation is based on size, charge and shape of
the protein (proteins are not denatured and
can potentially retain function or activity)
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SDS-Polyacrylamide Gel (cont)
Separation of proteins based on their size
is linear in relation to the distance migrated in
the gel. Using protein standards of known mass
and staining of the separated proteins with dye,
the mass of the proteins in the sample can be
determined. This is useful for purification and
diagnostic purposes.
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Gel filtration
Separation is based on protein size. Dextran or
polyacrylamide beads of uniform diameter are
manufactured with different pore sizes.
Depending on the sizes of the proteins to
be separated, they will enter the pore if small
enough, or be excluded if they are too large.
Hydrophobic Chromatography
Proteins are separated based on their net content
of hydrophobic amino acids. A hydrocarbon chain
of 4-16 carbons is the usual type of resin.
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Ion Exchange Chromatography
Separation of proteins based on the net charge of
their constituent amino acids. Different salt
concentrations can be used to elute the bound
proteins into tubes in a fraction collector. As
shown below, resins for binding () or (-)
charged proteins can be used
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Affinity Chromatography

• Based on the target proteins ability to bind a
  specific ligand, only proteins that bind to this
  ligand will be retained on the column bead. This
  is especially useful for immunoaffinity
  purification of proteins using specific
  antibodies for them.
• Example

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Protein Structure Methods

• The sequence of a protein (or peptide) is
  determined using sophisticated Mass Spectrometry
  procedures. The three dimensional structures of
  proteins are determined using X-ray
  crystallographic and NMR (nuclear magnetic
  resonance) spectroscopic methods.
• Protein sequence data banks useful for structural
  and sequence comparisons
• Please note that the new discipline termed
  Proteomics is evolving to incorporate
  cross-over analysis of sequence data banks, Mass
  Spec methodology, and living cells

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• THANK Q