Proteins
Amino Acids, Peptides and Proteins
Learning objectives
• Amino acids share a common structure
• R groups provide different...
Proteins
• Proteins serve many functions:
– 1.Structure: collagen and keratin are the chief
constituents of skin, bone, ha...
Proteins
– 6. Protection: blood clotting involves the protein
fibrinogen; the body used proteins called antibodies to
figh...
Amino Acids
•Have an alpha- carbon
attached to:
• an amino group
• carboxyl group

• a hydrogen
• an R group
Chirality of Amino Acids
• With the exception of glycine, all protein-derived
amino acids have at least one stereocenter (...
Each R group
determines the
properties of the amino
acid

R groups can be
polar, nonpolar,
acidic, basic

Hundreds of modi...
Each R group
determines the
properties of an amino
acid

R groups can be
polar, nonpolar,
acidic, basic
Proteins are made of 20 amino acids
amino acids can act as acids and bases

• Amino acids exist in solution as dipolar ions (Zwitterions)
• Like buffers, AA’s...
titration of amino acids
Ex. Glycine Deprotonation
• Two distinct
plateaus, each
correspond to
deprotonation of glycine

•...
proteins
formation of peptide bonds
Peptides and proteins are
polymers of amino acids
• Two amino acids are
covalently joined in
co...
Peptides: how aa are linked
• proteins are long chains of amino acids joined by amide
bonds.
peptide bond:
– amino acids b...
α-carbons separated by 3 covalent bonds
Partial sharing of
e-

• A small electric dipole results from the partial negative...
planar nature of peptide bonds

• The N-Cα and Cα-C bonds can rotate
Primary Structure
• Just how important is the exact amino acid
sequence?
– Human insulin consists of two polypeptide chain...
Primary Structure
– Vasopressin and oxytocin are both nonapeptides but
have quite different biological functions.

– Vasop...
proteins range in size
proteins contain prosthetic groups

Non-amino acid part of proteins
protein purification (chromatography)
Ion-Exchange

Size-Exclusion

Affinity

Uses protein characteristics, such as charge...
electrophoresis (SDS-PAGE)
Purification steps

Stain and
blot the gel

Migration of charged proteins in an electric field
...
2D gel electrophoresis

1. Isoelectric focusing

2. SDS PAGE
Mass Spectrometry
1. First treat isolated
protein with a protease
2. Mixture is vaporized
and peptides
separated
3. One pe...
Mass spectrometer
Mass Spectrum of Ethanol
Levels of Structure
• Primary structure: the sequence of amino acids
• Secondary structure: conformations of amino acids
i...
4 levels of protein structure

• Primary – sequence of amino acids
• Secondary – interactions between adjacent amino
acids...
Secondary Structure
• conformations of amino acids in localized regions
of a polypeptide chain.
– The most common types of...
-Helix
• The -helix structure: held together by hydrogen
bonds
-Helix
• In a section of -helix;
– The C=O group of each
peptide bond is hydrogen
bonded to the N-H group
of the peptide b...
secondary structure
• Note the position of the
purple R groups relative
to the backbone of the
polypeptide
all α helices are right handed
• But some
supramolecular
complexes are
left handed
(keratin, collage
n)

right-handed = cl...
β sheet secondary structure
• More extended
• H-bonds may occur between amino acids some
distance from one another
• Adjac...
β sheets require β turns
• One third of amino acids are in turns or loops
• Gly and Pro are frequently found in turns
-Pleated Sheet
• In a section of -pleated sheet;
– The C=O and N-H groups of peptide bonds from
adjacent chains point towa...
Pleated Sheet Structure of
Proteins
secondary structure and function
Tertiary Structure
• the overall conformation of an entire polypeptide
chain.
• Tertiary structure is stabilized in severa...
Cysteine
• The -SH (sulfhydryl) group of cysteine is easily
oxidized to an -S-S- (disulfide).
the permanent wave that isn’t
Heat
+

New S-S bonds
Tertiary Structure
• Forces that stabilize 3° structure of proteins
Tertiary Structures of Proteins
• the three dimensional shape of proteins that results
from further crosslinking, folding ...
protein structure

ribbon

mesh
surface contour

Sperm Whale
Myoglobin

ribbon + side chains

space-filling model
relative compactness of proteins

• Hypothetical chain length of a protein if it were to
appear either as an α helix or β ...
Common Motifs stable folding patterns in globular proteins
Common Motifs
Common Motifs

β microglobulin
Common Motifs
Common Motifs
Complex domain from simple motifs

immunoglobulin
quaternary structure
Quaternary Structure
• the arrangement of polypeptide chains into a
noncovalently bonded aggregation.
– The individual cha...
Hemoglobin
• The 4° structure of hemoglobin: made up of 4
subunits
Fibrous proteins: α keratin

• Evolved for strength (hair, wool, nails, claws, quills…)
• Right handed α helix
• Coiled-co...
Fibrous proteins: collagen

• Like keratin, collagen also evolved to provide strength
• Left-handed a chain (not an α heli...
Fibrous protein: silk

• Fibroin, the silk protein is in the β conformation
• Rich in Ala and Gly (for close packing)
• Mo...
Denaturation
• the process of destroying the native conformation
of a protein by chemical or physical means.
– Some denatu...
protein folding and misfolding
• Molecular chaperones
assist in protein folding
for many
• Interact with partially
folded ...
Protein Function
• Protein function often includes reversible binding
interactions with other molecules.
• Complementary i...
oxygen-binding proteins have a
heme prosthetic group
oxygen-binding proteins have a
heme prosthetic group

hemoglobin

http://www.youtube.com/watch?v=5LjLFrmKTSA&feature=relat...
protein-ligand interactions can be measured
association equilibrium: Ka = [PL] / [P] [L]
dissociation equilibrium: Kd = [P...
Hemoglobin

Binds O2 is a cooperative process.

Binding affinity of Hb for O2 is increased by the O2
saturation of the mol...
hemoglobin-O2 binding is influenced by pH
Hb, binds H+ and CO2 as well as
O2, but all at different sites.
Binding of H+ an...
hemoglobin-O2 binding allosterically
modulated by 2,3-bisphosphoglycerate

BPG reduces the affinity of
Hb for O2.
BPG bind...
immune responses are mediated by protein
interactions that distinguish self and non-self
Cellular immune response - T cell...
muscle contraction is also based on protein
interactions and conformational changes
Muscle contraction occurs by the
slidi...
1.

What 2 functional groups are present in all amino acids?

2.

Name the simplest amino acid. Is it a chiral molecule?

...
5.

What is meant by the primary, secondary and tertiary structures of proteins?

6.

What type of bonds are responsible f...
9. What is the effect of the following changes on the O2 affinity of hemoglobin?
a)

Drop in pH of blood plasma

a)

A dec...
Chapters 3,4,5
Chapters 3,4,5
Chapters 3,4,5
Chapters 3,4,5
Chapters 3,4,5
Chapters 3,4,5
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Chapters 3,4,5

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Chapters 3,4,5

  1. 1. Proteins
  2. 2. Amino Acids, Peptides and Proteins Learning objectives • Amino acids share a common structure • R groups provide different chemical properties • Amino acids can ionize in aqueous solutions • Proteins can be purified and studied in a variety of ways • Protein structure has four levels of organization • Sequence homology generally translates to shared function
  3. 3. Proteins • Proteins serve many functions: – 1.Structure: collagen and keratin are the chief constituents of skin, bone, hair, and nails. – 2. Catalysts: virtually all reactions in living systems are catalyzed by proteins called enzymes. – 3. Movement: muscles are made up of proteins called myosin and actin. – 4. Transport: hemoglobin transports oxygen from the lungs to cells; other proteins transport molecules across cell membranes. – 5. Hormones: many hormones are proteins, among them insulin, oxytocin, and human growth hormone.
  4. 4. Proteins – 6. Protection: blood clotting involves the protein fibrinogen; the body used proteins called antibodies to fight disease. – 7. Storage: casein in milk and ovalbumin in eggs store nutrients for newborn infants and birds; ferritin, a protein in the liver, stores iron. – 8. Regulation: certain proteins not only control the expression of genes, but also control when gene expression takes place.
  5. 5. Amino Acids •Have an alpha- carbon attached to: • an amino group • carboxyl group • a hydrogen • an R group
  6. 6. Chirality of Amino Acids • With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the carbon) and are chiral. – The vast majority of protein-derived amino acids have the L-configuration
  7. 7. Each R group determines the properties of the amino acid R groups can be polar, nonpolar, acidic, basic Hundreds of modified amino acids
  8. 8. Each R group determines the properties of an amino acid R groups can be polar, nonpolar, acidic, basic
  9. 9. Proteins are made of 20 amino acids
  10. 10. amino acids can act as acids and bases • Amino acids exist in solution as dipolar ions (Zwitterions) • Like buffers, AA’s can act as proton donors or acceptors – “Amphoteric” compounds or “amphoteric electrolytes”
  11. 11. titration of amino acids Ex. Glycine Deprotonation • Two distinct plateaus, each correspond to deprotonation of glycine • Titration curves can be used to predict AA charge at a given pH • The isoelectric point (pI) is the pH at 0 charge
  12. 12. proteins
  13. 13. formation of peptide bonds Peptides and proteins are polymers of amino acids • Two amino acids are covalently joined in condensation reaction N-terminal C-terminal
  14. 14. Peptides: how aa are linked • proteins are long chains of amino acids joined by amide bonds. peptide bond: – amino acids become linked together to form peptide bonds with the elimination of water – The reaction takes place between the -COOH of one amino acid and the -NH2
  15. 15. α-carbons separated by 3 covalent bonds Partial sharing of e- • A small electric dipole results from the partial negative charge on oxygen and the partial positive charge on nitrogen • The shared electrons result in some double bond character and the lack of rotation
  16. 16. planar nature of peptide bonds • The N-Cα and Cα-C bonds can rotate
  17. 17. Primary Structure • Just how important is the exact amino acid sequence? – Human insulin consists of two polypeptide chains having a total of 51 amino acids. – In the table are differences between four types of insulin. A Chain positions 8-9-10 B Chain position 30 Human Cow -Thr-Ser-Ile-Ala-Ser-Val- -Thr -Ala Hog Sheep -Thr-Ser-Ile-Ala-Gly-Val- -Ala -Ala
  18. 18. Primary Structure – Vasopressin and oxytocin are both nonapeptides but have quite different biological functions. – Vasopressin is an antidiuretic hormone. – Oxytocin affects contractions of the uterus in childbirth and the muscles of the breast that aid in the secretion of milk.
  19. 19. proteins range in size
  20. 20. proteins contain prosthetic groups Non-amino acid part of proteins
  21. 21. protein purification (chromatography) Ion-Exchange Size-Exclusion Affinity Uses protein characteristics, such as charge, size and binding affinity to separate the protein
  22. 22. electrophoresis (SDS-PAGE) Purification steps Stain and blot the gel Migration of charged proteins in an electric field Gel slows migration in proportion to mass
  23. 23. 2D gel electrophoresis 1. Isoelectric focusing 2. SDS PAGE
  24. 24. Mass Spectrometry 1. First treat isolated protein with a protease 2. Mixture is vaporized and peptides separated 3. One peptide is selected and further fragmented 4. MS measures m/z ratios for all the fragments
  25. 25. Mass spectrometer
  26. 26. Mass Spectrum of Ethanol
  27. 27. Levels of Structure • Primary structure: the sequence of amino acids • Secondary structure: conformations of amino acids in localized regions of a polypeptide chain; examples are -helix, -pleated sheet, and random coil. • Tertiary structure: the complete three-dimensional arrangement of atoms of a polypeptide chain. • Quaternary structure: the spatial relationship and interactions between subunits in a protein that has more than one polypeptide chain.
  28. 28. 4 levels of protein structure • Primary – sequence of amino acids • Secondary – interactions between adjacent amino acids • Tertiary – 3D folding of the polypeptide • Quaternary – arrangements of multiple polypeptides
  29. 29. Secondary Structure • conformations of amino acids in localized regions of a polypeptide chain. – The most common types of secondary structure are helix and -pleated sheet. -Helix: a type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral. -Pleated sheet: a type of secondary structure in which two polypeptide chains or sections of the same polypeptide chain align parallel to each other
  30. 30. -Helix • The -helix structure: held together by hydrogen bonds
  31. 31. -Helix • In a section of -helix; – The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it. – All R- groups point outward from the helix.
  32. 32. secondary structure • Note the position of the purple R groups relative to the backbone of the polypeptide
  33. 33. all α helices are right handed • But some supramolecular complexes are left handed (keratin, collage n) right-handed = clockwise
  34. 34. β sheet secondary structure • More extended • H-bonds may occur between amino acids some distance from one another • Adjacent chains can run parallel or anti-parallel to each other
  35. 35. β sheets require β turns • One third of amino acids are in turns or loops • Gly and Pro are frequently found in turns
  36. 36. -Pleated Sheet • In a section of -pleated sheet; – The C=O and N-H groups of peptide bonds from adjacent chains point toward each other so that hydrogen bonding is possible between them. – All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.
  37. 37. Pleated Sheet Structure of Proteins
  38. 38. secondary structure and function
  39. 39. Tertiary Structure • the overall conformation of an entire polypeptide chain. • Tertiary structure is stabilized in several ways: – Covalent bonds, as for example, the formation of disulfide bonds between cysteine side chains. – Hydrogen bonding between polar groups of side chains, as for example between the -OH groups of serine and threonine. – Electrostatic interaction or Salt bridges, as for example, the attraction of the -NH3+ group of lysine and the -COO- group of aspartic acid. – Hydrophobic interactions, as for example, between the nonpolar side chains of phenylalanine and isoleucine.
  40. 40. Cysteine • The -SH (sulfhydryl) group of cysteine is easily oxidized to an -S-S- (disulfide).
  41. 41. the permanent wave that isn’t Heat + New S-S bonds
  42. 42. Tertiary Structure • Forces that stabilize 3° structure of proteins
  43. 43. Tertiary Structures of Proteins • the three dimensional shape of proteins that results from further crosslinking, folding and interaction between R groups
  44. 44. protein structure ribbon mesh surface contour Sperm Whale Myoglobin ribbon + side chains space-filling model
  45. 45. relative compactness of proteins • Hypothetical chain length of a protein if it were to appear either as an α helix or β sheet
  46. 46. Common Motifs stable folding patterns in globular proteins
  47. 47. Common Motifs
  48. 48. Common Motifs β microglobulin
  49. 49. Common Motifs
  50. 50. Common Motifs
  51. 51. Complex domain from simple motifs immunoglobulin
  52. 52. quaternary structure
  53. 53. Quaternary Structure • the arrangement of polypeptide chains into a noncovalently bonded aggregation. – The individual chains are held together by hydrogen bonds, electrostatic interactions, and hydrophobic interactions. • Hemoglobin – Adult hemoglobin: two chains of 141 amino acids each, and two chains of 146 amino acids each. – Each chain surrounds an iron-containing heme unit.
  54. 54. Hemoglobin • The 4° structure of hemoglobin: made up of 4 subunits
  55. 55. Fibrous proteins: α keratin • Evolved for strength (hair, wool, nails, claws, quills…) • Right handed α helix • Coiled-coil provides added strength (like a twisted rope)
  56. 56. Fibrous proteins: collagen • Like keratin, collagen also evolved to provide strength • Left-handed a chain (not an α helix) • Right handed coiled coils – 3-stranded coil
  57. 57. Fibrous protein: silk • Fibroin, the silk protein is in the β conformation • Rich in Ala and Gly (for close packing) • More extended than α helix conformation
  58. 58. Denaturation • the process of destroying the native conformation of a protein by chemical or physical means. – Some denaturations are reversible, while others permanently damage the protein.
  59. 59. protein folding and misfolding • Molecular chaperones assist in protein folding for many • Interact with partially folded or improperly folded polypeptides • Misfolded proteins can be lethal Vacuoles associated with spongiform encephalopathies
  60. 60. Protein Function • Protein function often includes reversible binding interactions with other molecules. • Complementary interactions between proteins and ligands are the basis of self vs non-self recognition by the immune system. • Specific protein interactions modulated by chemical energy are the basis of muscle movement.
  61. 61. oxygen-binding proteins have a heme prosthetic group
  62. 62. oxygen-binding proteins have a heme prosthetic group hemoglobin http://www.youtube.com/watch?v=5LjLFrmKTSA&feature=related
  63. 63. protein-ligand interactions can be measured association equilibrium: Ka = [PL] / [P] [L] dissociation equilibrium: Kd = [P] [L] / [PL] O2 binding to myoglobin θ = fraction of ligand-binding sites occupied Which protein (X or Y) has greater affinity for ligand A?
  64. 64. Hemoglobin Binds O2 is a cooperative process. Binding affinity of Hb for O2 is increased by the O2 saturation of the molecule with the first O2 bound influencing the shape of the binding sites (conformation change) for the next O2
  65. 65. hemoglobin-O2 binding is influenced by pH Hb, binds H+ and CO2 as well as O2, but all at different sites. Binding of H+ and CO2 is inversely related to binding of O2. Low pH = high [H+] = lower O2 binding.
  66. 66. hemoglobin-O2 binding allosterically modulated by 2,3-bisphosphoglycerate BPG reduces the affinity of Hb for O2. BPG binds at a site distant from the O2-binding site and regulates the affinity of Hb for O2.
  67. 67. immune responses are mediated by protein interactions that distinguish self and non-self Cellular immune response - T cells destroy host cells infected by viruses Humoral immune response – B cells produce antibodies or immunoglobulins against bacteria, viruses and foreign molecules
  68. 68. muscle contraction is also based on protein interactions and conformational changes Muscle contraction occurs by the sliding of the thick (myosin) and thin (actin) filaments past each other Conformational changes in the myosin head are coupled to ATP hydrolysis http://www.sci.sdsu.edu/movies/actin_myosin _gif.html
  69. 69. 1. What 2 functional groups are present in all amino acids? 2. Name the simplest amino acid. Is it a chiral molecule? 3. Approximately how many amino acids are needed to make the proteins found in the body?
  70. 70. 5. What is meant by the primary, secondary and tertiary structures of proteins? 6. What type of bonds are responsible for the helix structure of some proteins? 5. Linus Pauling and Robert Corey found that the C—N bond in the peptide link is intermediate in length between a single and double bond. They also found that the peptide bond is planar. a) What does the length of the bond tell us about the strength and bond order? b) What does the observations tell us about the ease of rotation about the C—N peptide bond?
  71. 71. 9. What is the effect of the following changes on the O2 affinity of hemoglobin? a) Drop in pH of blood plasma a) A decrease of partial pressure of CO2 in the lungs a) Increase in BPG levels a) Increase in CO
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