3-d structure of proteins by doba jackson, ph.d
TRANSCRIPT
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3-D Structure of Proteins
By
Doba Jackson, Ph.D.
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Amino Acids Can Join Via Peptide Bonds
This reaction is thermodynamically unfavorable
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Peptide Bond, Facts
• Usually found in the trans conformation• It has (40%) double bond character• It is about 0.133 nm long –
• Single bond length: .120 nm• double bond length: .151 nm
• Six atoms of the peptide bond group are always planar! • N partially positive; O partially negative
Peptide Bond
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Peptide bond is rigid and Planar
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The language of Protein Chemists• Multisubunit- Proteins that have two or more
polypeptides attached non-covalently.– Oligomeric- Two of the same subunits associated.– Protomers- identical subunits of a multisubunit
protein.
• Prosthetic Group- a covalently attached non-amino acid part of a protein (cofactor, vitamins)
• Lipoproteins- proteins with a covalently attached lipid.
• Glycoproteins- proteins with covalently attached carbohydrates
• Metaloproteins- proteins that contain a specific metal atom attached
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“Peptides”• Short polymers of amino acids• 2, 3 residues – dipeptide, tripeptide• 12-20 residues - oligopeptide
What is this peptide sequence? S G Y A L
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Levels of Protein Structure
• Primary structure- A description of the covalent bonds linking amino acids in a peptide chain
• Secondary Structure- An arrangement of amino acids giving rise to structural patterns
• Tertiary Structure- Describes all aspects of three dimensional folding of a polypeptide
• Quarternary Structure- The arrangement in space of polypeptide units
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How do polypeptides fold in 3-D space
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Rules for protein folding
• Local amino acids fold upon each other in order to maximize number of hydrogen bonds produced (secondary structure).– α-helix– β-sheet– β-turn
• Globally, secondary structures fold upon each other in order to minimize the hydrophobic amino acid’s exposure to water (tertiary structure).
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Two angles along the α-carbon determine the secondary structure of a protein which are the
phi (ϕ) and psi (Ψ)
Ramachandran Plot
-Dark blue means most favorable conformation-Medium blue means less favorable conformation but still allowed-Light blue means the conformation is mildly strained-Yellow means the conformation is not allowed
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α-helix- Spiral arrangement- Phi = 60*- Psi = 45-50*- Pitch = 5.4 A- 3.6 residues per turn
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In an alpha-helix, all side chains extend perpendicular to the helical axis
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Amino acids 3 to 4 residues apart can have favorable interactions: ex.-Troponin C
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Left-handed helix
Right-handed helix
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Five types of constraints affect the stability of the alpha-helix
1- Electrostatic repulsion of successive amino acid residues
2- Bulkiness of adjacent R-groups
3- Interactions of R-groups spaced 3 to 4 residues apart
4- Presence of Proline or Glycine
5- Interactions of amino acid R-groups with the helical dipole
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Beta conformation organizes the polypeptide chain into sheets
- Beta sheet structures are nearly fully extended polypeptide chains
- Phi = -110 to -180*- Psi = +110 to +180*
- R-groups extend in protruding opposite directions from the polypeptide chains
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Antiparalell Beta-sheet structure can a maximum overlap of H-bonds
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Parallel Beta-sheet structure is less stable and only has weakly overlapping H-bonds
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- Short turns are characterized by a H-bond between the first and third AA.
- Different turns are characterized by the dihedral angles
- Often turns are between two strands of anti-parallel beta sheets
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- Most but not all residues fall within the allowed regions
- Glycines many times falls outside the allowed regions because it is the least sterically hindered
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Globular proteins versus Fibrous proteins
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• Alpha-Keratin (Hair, Nails)- has high tensile strength and is water insoluble.
• Collagen (Cartilage, Tendons, Ligaments, Skin, Blood Vessels)- Has high tensile strength (less than keratin) and water soluble.
• Silk (spider web)- smooth and low strength
Fibrous proteins are adapted for a structural function
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Structure of Keratin (Hair) illustrates high strength of the helical structure
Acidic Keratin (green)Basic Keratin (grey)
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α-Keratin forms a two-stranded Coiled Coil Structure
Helix-Wheel Representation
a, d, a’, d’ are hydrophobic residues
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Structure of Collagen (cartilage) illustrates high strength and flexibility
Repeated structure of Gly-X-Proline
Triple helix of Collagen
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Structure of Collagen
Right-Handed or Left-Handed?
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Structure of Collagen
• Every third residue must be Glycine due to steric crowding within the triple helix
• Prolyl Hydroxylase adds hydroxyl groups to 3’ and 4’ positions of proline
• The hydroxyl group add stability to collagen by intramolecular hydrogen bonding.
• Prolyl Hydroxlase utilizes ascorbic acid (vitamin C) as a cofactor. Lack of vitamin C causes (Scurvy) causes skin lesions, fragile blood vessels, and poor wound healing.
Type I Collagen SequenceKey points to Collagen structure
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Structure of Collagen
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Globular proteins versus Fibrous proteins
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Experimental Methods Used to Determine Macromolecular Structures
• X-ray Crystallography- A technique that directly images molecules using X-rays.
• NMR- Spectroscopy- A technique that determines a protein structure based on distance restraints determined from coupling of nuclei either through space (NOESY) or through bonds (COSY).
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X-ray crystallography is how we determine structures of most proteins
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NMR spectroscopy is how we determine structures of other proteins
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How to view protein structures
Ribbon Diagram Mesh Diagram Mesh Diagram
Ribbon Diagram /w side chains
Surface DiagramUsing van der Waals radii
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General Properties of Globular Tertiary Structures
• Tertiary Structure- the folding of 2º structure elements and spacial position of the side chains
• Side chains are arranged according to polarity:– Nonpolar residues: Val, Leu, Ile, Met, Phe, Trp, Tyr
are mainly found in the interior away from water– Polar Charged residues: Arg, Lys, Asp, Glu: are
mainly found on the surface of proteins– Polar Neutral residues: Ser, Thr, Gln, Asn, are
usually on the exterior but often found in the interior.
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X-ray Structure of Horse Heart Cytochrome C
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X-ray Structure of Horse Heart Cytochrome C
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General Terms of Globular Tertiary Structures
• Structural Families (Folds): Proteins that have similar tertiary structures are considered to belong to the same family – Globin Family (Hemoglobin, Myoglobin, etc)– Rossmann Fold (Dehydrogenases, etc)
• Domains- A single isolatable tertiary structure with a hydrophobic core
• Motifs- a small building block of a tertiary structure (or domain)
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Some Known Motifs
βαβ -motif β- hairpin motif αα -motif
Greek Key-motif
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The α/β Barrel family has a βαβ-motif as a fundamental unit
Triose Phosphate Isomerase
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Quaternary Structure
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Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8Ǻ
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Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8Ǻ
Nonpolar residues
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Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8ǺCharged polar residues
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Protein Structure vs Function
• Proteins have active sites– Ligand binding sites– Catalytic sites (enzymes)– Regulatory sites
• Proteins have dynamic and flexible conformations– Induced fit- conformations change upon ligand
binding– Cooperativity- multiple active site can coordinate
their activities.
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Globular Tertiary Structures
• Protein Families: Proteins that have similar primary sequences are considered belonging to the same family – Globin Family (Hemoglobin, Myoglobin, etc)
– Rossmann Fold (Dehydrogenases, etc)
• Domains- A single isolatable tertiary structure with a hydrophobic core
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Lets consider oxygen transport proteins
Problem:
- Oxygen lacks a dipole moment.
- Oxygen has low solubility in water.
- Oxygen doesn’t bind any of the amino acids
Nature’s Solution
- Use transition metals (Fe, Cu) to coordinate with oxygen’s
lone pairs.
OO
Lone Pairs
Fe
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Oxygen Transport
Nature’s Solution
- We can prevent any reactivity of the iron-oxygen complex by blocking all
of the other 5 coordination sites on Fe.
OO
Fe
Another Problem
- Transition metals (Fe, Cu) will react with oxygen to from free radicles.
Plane (Porphyrin ring)
Protein
What is the Fe-O-O angle?
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Hemoglobin: Protein Function in a Microcosm
By Doba Jackson
Assistant Professor of Chemistry & BiochemistryHuntingdon College
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Iron-Porphyrin complex (Heme)
Pyrole Pyrole
Pyrole Pyrole
Proprionate
Vinyl
Vinyl
Methyl Methyl
Methyl
Methyl
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Characteristics of Myoglobin
- Myoglobin is a protein which binds oxygen in red muscle (heart, skeletal muscle).
- Cells without myoglobin depend on the supply of oxygen from red blood cells (hemoglobin).
- Myoglobin is a single polypeptide of ~ 150 amino acids and 8 α-helical segments
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Heme is inside a hydrophobic
interior
Two Proprionates of Heme are surface
assessable
Proximal His occupies the 5th coordination
site of Fe
Oxygen
ProteinOxygenNitrogenCarbon
Structure of Myoglobin
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Distal His coordinates To the second oxygen
Proximal His
ProteinOxygenNitrogenCarbon
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Introduction to Hemoglobin
• Hemoglobin is the oxygen carrying protein in red blood cells.
• Hemoglobin makes up 97% (+ bound water) of the red blood cell contents.
• Hemoglobin consist of 4 polypeptides arranged as a tetramer.
• (2) α-subunits (α1 and α2)• (2) β-subunits (β1 and β2)
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Quiz 3 (25 pts) • Go to Jmol Protein Explorer frontdoor:
– http://chemapps.stolaf.edu/pe/protexpl/htm/index.htm
• Type in 1HGA (PDB ID for T-state) • Color as you wish• Take a picture (edit-copy-paste to Word )• Do the same for 1BBB (PDB ID for R-state )• Write a paragraph convincing me that these are
unique structures.
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Both the α and β subunits are structurally similar to myoglobin
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Mb Hbα Hbβ Mb Hbα Hbβ Mb Hbα Hbβ
29 of 141 amino acid residues are the exact same in human Myoglobin (Mb), Hemoglobin α (Hbα), Hemoglobin β (Hbβ)
Proximal Histidine
Distal Histidine
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Structure of Hemoglobin demonstrates symmetry in its quaternary structure
α Subunit
α Subunit
β Subunit
β Subunit
Two-fold axis
Two-fold axis
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Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
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Quantitative description of Myoglobin-Oxygen Binding
2 2
2
2
2
2
2
2 2
tan
1tan
Rearrange the association equation to solve for [MbO ]
Fraction of ligand binding to protein is
Bind
A A
D DA
A
Mb O MbO
MbOK K association cons t
Mb O
Mb OK K dissociation cons t
MbO K
K Mb O MbO
2
2
2 2 2 2
2 2 22
ing sites occupied
Total binding sites
11A A
A A D
A
MbO
MbO Mb
K Mb O K O O O
K Mb O Mb K O O KOK
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2 2
tan
1tan
Rearrange the association equation to solve for [PL]
Fraction of ligand binding to protein is
Binding sites occu
A
D
K association cons tA
K dissociation cons tD K A
P L PL
PLK
P L
P LK
PL
K Mb O MbOA
2
2
2 2 2 2
2 2 22
pied
Total binding sites
11A A
A A D
A
MbO
MbO Mb
K Mb O K O O O
K Mb O Mb K O O KOK
Previous Slide
2 2
2
2
2
2
2
2 2
tan
1tan
Rearrange the association equation to solve for [MbO ]
Fraction of ligand binding to protein is
Bind
A A
D DA
A
Mb O MbO
MbOK K association cons t
Mb O
Mb OK K dissociation cons t
MbO K
K Mb O MbO
2
2
2 2 2 2
2 2 22
ing sites occupied
Total binding sites
11A A
A A D
A
MbO
MbO Mb
K Mb O K O O O
K Mb O Mb K O O KOK
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θ =
A Hyperbola!!!!
Special Case: θ = .5 (or ½)
2
2
2 2
2 2
2
2
2 50
50
50
50
1
2
2
2
o
D O
O O
O O
O
PO
O K P P
P P P
P P P
P P
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Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
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Quantitative description of Hemoglobin binding to Oxygen
2
2
2
2
2 2
2
2
2 2
2
50
50
50
50
50
1 1
? ?1
1
n
nA n
DO
n
O
n
O
n
O
n n
O O
n
O
n
O
Hb nO Hb O
Hb OK
K PHb P
P
P P
P
P P P
PP
P P
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2
2
2
50
50
50
1
1
1
n
O
n
O
O
P
P
PLog Log
P
Log n Log P Log P
Quantitative description of Hemoglobin binding to Oxygen
n = slopeLog P50 = intercept
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Hill plot (Archibald Hill, 1910)
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(linear)Low oxygen affinity
(Hyperbolic) High oxygen affinity
Hemoglobin (sigmodial)
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Hemoglobin is an Allosteric protein and Myoglobin is not
• Allosteric Protein- A protein in which the binding of a ligand to one site effects the binding properties of another site on the same protein.
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Hill constant (NH) is a measure of cooperativity
NH = 1 No Cooperativity
NH > 1 Positive Cooperativity
NH < 1 Negative Cooperativity
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Hemoglobin undergoes a structural change when it binds to oxygen
Tense State (T-state) Relaxed State (R-state)
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Lysine 40α-chain
His 149β-chain
Aspartate 93β-chain
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Electrostatic interactions stabilize the T-state of Hemoglobin
Lysine 40α-chain
His 149β-chain Aspartate 93
β-chain
Asp His
93 149
His Asp
149 93
Lys
40
PDB ID: 1HGA
Lys
40
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His 149β1-chain
His 149β2-chain
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Electrostatic interactions stabilize the T-state of Hemoglobin
PDB ID: 1BBB
His 149β1-chain
Asp His
93 149
His Asp
149 93
Lys
40
Lys
40
His 149β2-chain
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Oxygen binding triggers a conformational change from T-state to R-state
No OxygenT-state
No OxygenR-state
60 pm puckerValine
Leucine
Leucine
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Summarize the conformational change of Hemoglobin
• Hemoglobin undergoes a conformational change from the T-state to the R-state
• Oxygen binding stimulates the conversion from the T-state to the R-state.
• The T-state is stabilized by many ionic interactions that are not present in the R-state (ex. His 146).
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Summarize the conformational change of Hemoglobin
• The center cavity of hemoglobin becomes narrower.
• The center of the Fe atom is 60 pm below the porphyrin ring in the T-state but not in the R-state.
• Hydrophobic interactions between the protein and the of the porphyrin ring are stronger in the R-state.
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Problem #2: Which of the following situations would produce a Hill Plot
with NH <1
A)The protein has multiple binding sites each with a single ligand-binding site. The binding to one site decreases the affinity of binding to the other sites.
Yes or No?
Yes
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Problem #2: Which of the following situations would produce a Hill Plot
with NH <1
B) The protein is a single polypeptide with two ligand binding sites each having a different affinity for
ligand.
Yes or No?
Yes
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Problem #2: Which of the following situations would produce a Hill Plot
with NH <1
C) The protein has a single polypeptide with one ligand binding site. When purified, the protein preparation is heterogeneous and has some of the molecules
inactive.
Yes or No?
Yes
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Concerted Model
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Sequential Model
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Blood, extracellular Fluid
Lungs, Air space
How does CO2 fit in?
(H+)-Hb Hb + H+
(H+)-Hb Hb + O2
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Effect of pH on the binding of oxygen to Hemoglobin
Lungs
Tissues
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Normal Red Blood Cells
Sickle-Cell Anemia Red Blood Cells
Substitution of a Valine for a Glutamic acid on the surface of Hemoglobin β-subunit is the cause
of Sickle-Cell Anemia
V-
Hydrophobic patch
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Protein Function II: The Immune System
By Doba Jackson, Ph.D.
Associate Professor of Chemistry and BiochemistryHuntingdon College
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Complementary interactions: The Immune system
• Humoral Immune System- uses membrane bound and secreted antibodies from B-Lymphocytes directed toward bacteria and foreign proteins. Most effective for bacterial and viral infections.
B- LymphocytesT- helper cells (Th cells)Major Histocompatability Complex (MHC)
• Cellular Immune System- uses receptors on the surface of T-Lymphocytes to recognize whether a cell has been invaded by a foreign host.
• Cytotoxic T-lymphocytes (Tc cells)• T-helper cells• T-memory cells• Major Histocompatability Complex (MHC)
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Important LymphocytesLymphocytes are distinguished by having a deeply staining nucleus that may be
eccentric in location, and a relatively small amount of cytoplasm. Lymphocytes are common in the blood and lymphatic system.– B cells make antibodies that can bind to pathogens, block pathogen invasion,
activate the complement system, and enhance pathogen destruction.– T cells have multiple roles:
• CD4+ helper T cells: T cells displaying co-receptor CD4 are known as CD4+ T cells. These cells have T-cell receptors and CD4 molecules that, in combination, bind antigenic peptides presented on major histocompatibility complex (MHC) class II molecules on antigen-presenting cells. Helper T cells make cytokines and perform other functions that help coordinate the immune response.
• CD8+ cytotoxic T cells: T cells displaying co-receptor CD8 are known as CD8+ T cells. These cells bind antigens presented on MHC I complex of virus-infected or tumor cells and kill them.
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The Complex Immune System
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Cellular Immune System
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Major Histocompatability Complexes (MHC’s) is essential to the Cellular Immune
System
- Both MHCs have both an α and β chainshowever, the class I MHC protein has a small non-membrane spanning β chain
whereas the β-chain of class II MHC protein has two membrane spanning β-
chain.
- Class I MHC proteins are found on the surface of virtually all vertebrate cells.
- Class II MHC proteins occur on a few types of specialized cells that include
macrophages and B-cells.
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The Complex Immune System
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Helper T-cells activation
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B-cells are activated using cell surface antibodies and T-helper cells
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Class I MHC protein
- Typical cellular proteins are digested inside the cell by proteases
then each peptide is displayed by MHC proteins.
- T- cell receptors recognize the MHCproteins with the bound antigen. If the
bound antigen is foreign, the T-cell receptor will lyse the cell and dispense
its contents.
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Humoral Immune System uses immunoglobulins (antibodies)
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Memory T-cells and B-cells improve immune response upon secondary
exposure to antigen
Normal lymphocytes live 1 to 2 days but memory T and B cells can live for decades.
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Recognition of the Antibody-Antigen Complex
In order to generate an optimal fit for the antigen, the variable domains of
the antibody will often undergo a slight conformational change.
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Different Immunoglobulin subtypes occur in all B-cells
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The Immune System is Self-Tolerant
• Self-tolerance is developed during pregnancy period where protein digests of its own self are displayed by the MHC complex and generates memory T and B-cells. These cells are destroyed upon birth.
• Occasionally, the immune system attacks its own antigen after the selection period. This results in autoimmune diseases.
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Antibodies develop high affinity for binding foreign antigen sites within the
variable domains
• The binding specificity is determined by the amino acids located on the variable domains of heavy and light chains.
• Specificity is conferred by chemical complementarities between the antigen and its specific binding site in terms of molecular shape and location of charged, nonpolar, and hydrogen bonding groups.
• Typical antigen-antibody interactions are strong with Kd values that are as low as 10-10 M.
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Induced fit in the binding of IgG to an Antigen
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The high affinity and specificity of antibodies make them very useful for biological assays
ELISA Enzyme-linked immunosorbant assay
Western Blot
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Protein Function III: Muscle Contraction
By Doba Jackson, Ph.D.
Associate Professor of Chemistry and BiochemistryHuntingdon College
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Myosin has a globular amino terminus and a long coiled coil tail
17 nm Head
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Myosin, Actin Filaments
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Striated Muscle Fibers
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