4 Proteins

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4 Proteins
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Protein Structure

• 1 structure the sequence of amino acids in a
  polypeptide chain, read from the N-terminal end
  to the C-terminal end
• 2 structure the ordered 3-dimensional
  arrangements (conformations) in localized regions
  of a polypeptide chain refers only to
  interactions of the peptide backbone
• e. g., ?-helix and ?-pleated sheet

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Ramachandran Angles
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?-Helix
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?-Helix

• coil of the helix is clockwise or right-handed
• there are 3.6 amino acids per turn
• repeat distance is 5.4Å
• each peptide bond is s-trans and planar
• CO of each peptide bond is hydrogen bonded to
  the N-H of the fourth amino acid away
• CO----H-N hydrogen bonds are parallel to helical
  axis
• all R groups point outward from helix

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?-Helix

• Several factors can disrupt an ?-helix
• proline creates a bend because of (1) the
  restricted rotation due to its cyclic structure
  and (2) its ?-amino group has no N-H for hydrogen
  bonding
• strong electrostatic repulsion caused by the
  proximity of several side chains of like charge,
  e.g., Lys and Arg or Glu and Asp
• steric crowding caused by the proximity of bulky
  side chains, e.g., Val, Ile, Thr

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?-Pleated Sheet
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?-Pleated Sheet

• polypeptide chains lie adjacent to one another
  may be parallel or antiparallel
• R groups alternate, first above and then below
  plane
• each peptide bond is s-trans and planar
• CO and N-H groups of each peptide bond are
  perpendicular to axis of the sheet
• CO---H-N hydrogen bonds are between adjacent
  sheets and perpendicular to the direction of the
  sheet

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? -TURN
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?-Helices and ?-Sheets

• Supersecondary structures the combination of ?-
  and ?-sections, as for example
• ??b unit two parallel strands of ?-sheet
  connected by a stretch of ?-helix
• ?? unit two antiparallel ?-helices
• ?-meander an antiparallel sheet formed by a
  series of tight reverse turns connecting
  stretches of a polypeptide chain
• Greek key a repetitive supersecondary structure
  formed when an antiparallel sheet doubles back on
  itself
• ?-barrel created when ?-sheets are extensive
  enough to fold back on themselves

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Collagen Triple Helix

• consists of three polypeptide chains wrapped
  around each other in a ropelike twist to form a
  triple helix called tropocollagen MW approx.
  300,000
• 30 of amino acids in each chain are Pro and Hyp
  (hydroxyproline) hydroxylysine also occurs
• every third position is Gly and repeating
  sequences are X-Pro-Gly and X-Hyp-Gly
• each polypeptide chain is a helix but not an
  a-helix
• the three strands are held together by hydrogen
  bonding involving hydroxyproline and
  hydroxylysine
• with age, collagen helices become cross linked by
  covalent bonds formed between Lys and His
  residues
• deficiency of Hyp results in fragile collagen

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Collagen Triple Helix
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Factors Determining Secondary and Tertiary
Structure
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3 and 4 Structure

• Tertiary (3) structure the arrangement in space
  of all atoms in a polypeptide chain
• it is not always possible to draw a clear
  distinction between 2 and 3 structure
• Quaternary (4) structure the association of
  polypeptide chains into aggregations
• Proteins are divided into two large classes based
  on their three-dimensional structure
• fibrous proteins
• globular proteins

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Fibrous Proteins

• Fibrous proteins contain polypeptide chains
  organized approximately parallel along a single
  axis. They
• consist of long fibers or large sheets
• tend to be mechanically strong
• are insoluble in water and dilute salt solutions
• play important structural roles in nature
• Examples are
• keratin of hair and wool
• collagen of connective tissue of animals
  including cartilage, bones, teeth, skin, and
  blood vessels

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keratins
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Globular Proteins

• Globular proteins proteins which are folded to a
  more or less spherical shape
• they tend to be soluble in water and salt
  solutions
• most of their polar side chains are on the
  outside and interact with the aqueous environment
  by hydrogen bonding and ion-dipole interactions
• most of their nonpolar side chains are buried
  inside
• nearly all have substantial sections of ?-helix
  and ?-sheet
• Examples are
• myoglobin (Figure,6-31)
• hemoglobin

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Factors Directing Folding

• Noncovalent interactions, including
• hydrogen bonding between polar side chains, e.g.,
  Ser and Thr
• hydrophobic interaction between nonpolar side
  chains, e.g., Val and Ile
• electrostatic attraction between side chains of
  opposite charge, e.g., Lys and Glu
• electrostatic repulsion between side chains of
  like charge, e.g., Lys and Arg, Glu and Asp
• Formation of disulfide (-S-S-) bonds between side
  chains of cysteines

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Factors Determining Tertiary Structure
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-S-S-

• BPTI

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3 Structure

• x-ray crystallography
• uses a perfect crystal that is, one in which all
  individual protein molecules have the same 3D
  structure and orientation
• exposure to a beam of x-rays gives a series
  diffraction patterns
• information on molecular coordinates is extracted
  by a mathematical analysis called a Fourier
  series
• 2-D Nuclear magnetic resonance
• can be done on protein samples in aqueous solution

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Quaternary Structure

• Quaternary (4) structure the association of
  polypepetide monomers into multisubunit proteins
• examples we will see in this course

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Denaturation

• Denaturation the loss of the structural order
  (2, 3, 4, or a combination of these) that
  gives a protein its biological activity that is,
  the loss of biological activity
• Denaturation can be brought about by
• heat
• large changes in pH, which alter charges on side
  chains, e.g., -COO- to -COOH or -NH? to -NH?
• detergents such as sodium dodecyl sulfate (SDS)
  which disrupt hydrophobic interactions
• urea or guanidine, which disrupt hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds

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Denaturation
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Protein Folding Chaperones

• primary structure conveys all information
  necessary to produce the correct 3 structure
• nonetheless, in the protein-dense environment of
  a cell, proteins may begin to fold incorrectly or
  may associate with other proteins before folding
  is completed
• special proteins called chaperones aid in the
  correct and timely folding of many proteins
• hsp70 were the first protein chaperone discovered
• chaperones exist in organisms from prokaryotes to
  humans

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chaperone
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• Hemoglobin
• and
• Immunoglobulins

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Myoglobin
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Structure of Myoglobin

• a single polypeptide chain of 153 amino acids
• a single heme group in a hydrophobic pocket
• 8 regions of ?-helix no regions of ?-sheet
• most polar side chains are on the surface
• nonpolar side chains are folded to the interior
• two His side chains are in the interior, involved
  with interaction with the heme group
• Fe(II) of heme has 6 coordinates sites 4
  interact with N atoms of heme, 1 with N of a His
  side chain, and 1 with either an O2 molecule or
  an N of the second His side chain

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Heme structure

• P49 Figure 7-4

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Hemoglobin
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Oxygen Binding of Hb

• a tetramer of two ?-chains (141 amino acids each)
  and two ?-chains (153 amino acids each) a2b2
• each chain has 1 heme group hemoglobin can bind
  up to 4 molecules of O2
• binding is cooperative when one O2 is bound, it
  becomes easier for the next O2 to bind
• the function of hemoglobin is to transport oxygen
• the structure of oxygenated Hb is different from
  that of unoxygenated Hb
• H, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG)
  affect the ability of Hb to bind and transport
  oxygen

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Oxygen Binding of Hb

• P51 Fig7-8 O2 binding of hemoglobin and
  myoglobin
• Cooperativity of Binding/Release
• The oxygenation state (filled or empty) of one
  site of the multisubunit hemoglobin can be
  communicated to another site, resulting in
  cooperative binding and release of oxygen.
• Allosteric binding

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Oxygen Binding of Hb

• The effect of pH on the oxygen-binding ability of
  Hb is called the Bohr effect (p53 fig7-16)
• as pH decreases (more acidic), oxygen is released
• CO2 promotes release of O2 from HbO2

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Oxygen Binding of Hb

• Table Summary of the Bohr effect

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Hemoglobin (Hb)

• Hemoglobin in blood is bound to BPG
• interaction is electrostatic, between negative
  charges on BPG(2,3-bisphosphoglycerate
• and positive side chains (e.g., Lys, Arg) of
  hemoglobin
• BPG promotes O2 dissociation
• Hb stripped of BPG remains saturated with O2

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Fetal Hemoglobin, Hb F

• has a higher affinity for O2 than maternal Hb A
• structure is a2g2
• binds less strongly to BPG that does Hb A
• Figure Oxygen binding capacity of Hb F

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Abnormal Human Hb( Hb Variants)

• Hb S substitution of Val for Glu at 26b
• Hb E Glu B8(26)b -gt Lys change is on the
  surface and has little effect on Hb stability or
  function
• Hb Savannah Gly B6(24)b -gt Val not enough room
  for Val between B-helix and E-helix which
  disrupts entire structure
• Hb Bibba Leu H19(136)a -gt Pro proline disrupts
  the H-helix
• Hb M Iwate His F8(87)a -gt Tyr blood contains
  methemoglobin and blood is chocolate brown
• Hb Milwaukee Val E11(67)b -gt Glu glutamate side
  chain forms an ion pair with heme iron which
  stabilizes Fe(III) and prevents O2 binding

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Evolution of Myoglobin / Hemoglobin Proteins

• Out of the 153 amino acids in the amino acid
  sequences of sperm whale myoglobin and human
  myoglobin, there are only 25 differences.(?100
  million years)
• Conserved Amino Acid Sequences - During the long
  evolution of the myoglobin/hemoglobin family of
  proteins, only a few amino acid residues have
  remained invariant .

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Immuglobulins

• Antigens and Antibodies - The foreign substance
  that elicits an immune response is called the
  antigen. A specific immunoglobulin that binds to
  the antigen is called the antibody.
• 1.Humoral immune response - Lymphatic cells
  called B lymphocytes synthesize specific
  immunoglobulin molecules that are excreted from
  the cell and bind to the invading substance.
  Binding either precipitates the foreign substance
  or marks it for destruction by cells called
  macrophages.
• 2. Cellular immune response - Lymphatic cells
  called T lymphocytes, bearing immunoglobulin-like
  molecules on their surfaces, recognize and kill
  foreign or aberrant cells.

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Terms

• Antigen Foreign material that is recognized by
  the immune system, it is usually a protein, but
  it can be a peptide, or carbohydrate.
• Epitope Region of a protein antigen to which the
  antibody binds.
• Hapten A small chemical that is an antigen.

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Antibody Structure

• 1 Quaternary structure (2 Light 2 Heavy
  chains). The two heavy and light chains are held
  together by non-covalent forces and covalent
  (disulfide) bonds. The light chain consists of
  two immunoglobulin folds and the heavy chain
  contains four of these domains The overall shape
  is that of a Y. Two antigen binding
  sites/antibody.

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Antibody structure

• 2 Immunoglobulin fold is an example of a protein
  domain or a motif. It contains 7 b -strands that
  form a two sheet sandwich with 4 stands on one
  side and 3 on the other. A buried disulfide bond
  crosslinks the two faces.

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Antibody structure
CDR
CDR

• 3 Disulfide bonds covalently join the heavy and
  light chains, conferring stability on this
  secreted protein (Some antibodies are secreted
  outside the body)
• 4 hypervariable regions(CDR (Complementary
  determining region))
• The first immunoglobulin domain of the heavy and
  light chain contains three special segments of
  primary sequence that vary in their primary
  sequence from one antibody to the next.

• 5 In the folded form of the antibody the three
  hypervariable regions of each chain come together
  in space to form the binding site for foreign
  material.

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Antibody structure

• Fab fragments can be further reduced to Fv
  fragments, consisting of the 1st immunoglobulin
  fold from the heavy and light chain. The Fv
  domain is the smallest unit that can bind
  antigen.

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Practical Uses of Immunoglobulins

• Fluorescence tagging (to label various components
  in the cell)
• Purification of materials (More on this later)
• Immunotherapy (see Campbell)
• Novel chemical reactions (Some antibodies can
  actually perform chemical reactions)
• Drug detoxification, see Chime page on Antibodies
  and Angel dust (PCP)