Transcript Hemoglobin - Huntingdon College

3-D Structure of Proteins
By
Doba Jackson, Ph.D.
Amino Acids Can Join Via Peptide Bonds
This reaction is thermodynamically unfavorable
Peptide Bond, Facts
Peptide
Bond
• 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 is rigid and Planar
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
“Peptides”
• Short polymers of amino acids
• 2, 3 residues – dipeptide, tripeptide
• 12-20 residues - oligopeptide
What is this peptide sequence?
SGYAL
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
How do polypeptides fold in 3-D space
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).
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
α-helix
- Spiral arrangement
- Phi = 60*
- Psi = 45-50*
- Pitch = 5.4 A
- 3.6 residues per turn
In an alpha-helix, all side chains extend
perpendicular to the helical axis
Amino acids 3 to 4 residues apart can have
favorable interactions: ex.-Troponin C
Left-handed
helix
Right-handed
helix
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
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
Antiparalell Beta-sheet structure can
a maximum overlap of H-bonds
Parallel Beta-sheet structure is less stable
and only has weakly overlapping H-bonds
- 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
- 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
Globular proteins versus Fibrous
proteins
Fibrous proteins are adapted
for a structural function
• 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
Structure of Keratin (Hair) illustrates
high strength of the helical structure
Acidic Keratin (green)
Basic Keratin (grey)
α-Keratin forms a two-stranded
Coiled Coil Structure
Helix-Wheel Representation
a, d, a’, d’ are hydrophobic residues
Structure of Collagen (cartilage)
illustrates high strength and flexibility
Triple helix of Collagen
Repeated structure of Gly-X-Proline
Structure of Collagen
Right-Handed or Left-Handed?
Structure of Collagen
Key points to Collagen structure
• 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 Sequence
Structure of Collagen
Globular proteins versus Fibrous
proteins
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).
X-ray crystallography is how we determine
structures of most proteins
NMR spectroscopy is how we determine
structures of other proteins
How to view protein structures
Ribbon Diagram
Ribbon Diagram
/w side chains
Mesh Diagram
Surface Diagram
Using van der Waals radii
Mesh Diagram
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.
X-ray Structure of Horse Heart Cytochrome C
X-ray Structure of Horse Heart Cytochrome C
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)
Some Known Motifs
βαβ -motif
β- hairpin motif
αα -motif
Greek Key
-motif
The α/β Barrel family has a βαβmotif as a fundamental unit
Triose
Phosphate
Isomerase
Quaternary Structure
Structure of Horse Heart Cytochrome C
PDB ID: 3CYT
Res: 1.8Ǻ
Structure of Horse Heart Cytochrome C
PDB ID: 3CYT
Res: 1.8Ǻ
Nonpolar residues
Charged polar residues
Structure of Horse Heart Cytochrome C
PDB ID: 3CYT
Res: 1.8Ǻ
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.
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
Lets consider oxygen transport
proteins
Problem:
- Oxygen
Lone Pairs
lacks a dipole moment.
O
- Oxygen has low solubility in water.
- Oxygen
doesn’t bind any of the
amino acids
O
Fe
Nature’s Solution
- Use transition metals (Fe, Cu) to coordinate with oxygen’s
lone pairs.
Oxygen Transport
What is the Fe-O-O angle?
Another Problem
O
- Transition metals (Fe, Cu) will react
with oxygen to from free radicles.
Fe
Nature’s Solution
Protein
- We can prevent any reactivity of the
iron-oxygen complex by blocking all
of the other 5 coordination sites on Fe.
O
Plane (Porphyrin ring)
Hemoglobin: Protein Function in
a Microcosm
By
Doba Jackson
Assistant Professor of Chemistry & Biochemistry
Huntingdon College
Iron-Porphyrin complex (Heme)
Proprionate
Methyl
Pyrole
Pyrole
Methyl
Methyl
Vinyl
Pyrole
Pyrole
Methyl
Vinyl
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
Protein
Oxygen
Nitrogen
Carbon
Structure of Myoglobin
Oxygen
Two Proprionates
of Heme are surface
assessable
Proximal His occupies
the 5th coordination
site of Fe
Heme is inside a
hydrophobic
interior
Protein
Oxygen
Nitrogen
Carbon
Distal His coordinates
To the second oxygen
Proximal His
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)
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.
Both the α and β subunits are structurally
similar to myoglobin
29 of 141 amino acid residues are the exact same in human
Myoglobin (Mb), Hemoglobin α (Hbα), Hemoglobin β (Hbβ)
Mb Hbα Hbβ
Mb Hbα Hbβ
Mb Hbα Hbβ
Proximal
Histidine
Distal
Histidine
Structure of Hemoglobin demonstrates
symmetry in its quaternary structure
α Subunit
Two-fold
axis
β Subunit
Two-fold
axis
β Subunit
α Subunit
Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
Quantitative description of MyoglobinOxygen Binding

 MbO2
Mb  O2 

MbO2 

KA 
 MbO2 
Mb O2 

KD 
 MbO2 
K A  association constant
1
KD 
 dissociation constant
KA
Rearrange the association equation to solve for [MbO 2 ]
K A  Mb O2    MbO2 
Fraction of ligand binding to protein is 
KD 
 P L
 PL
Previous Slide
KD 
1

 MbO2
Mb  O2 

 dissociation
cons
 MbO
 tanK t  association constant
K 
2
 MbO2  A
 MbO2  K  1  dissociation constant
KD 
D
KA
Rearrange the association equation to solve MbO
for 2[PL]

KA
K  MbO2    MbO2 
A
A
Rearrange the association equation to solve for [MbO 2 ]
K A  MbO2    MbO2 
Fraction of ligand binding to protein is 
Fraction of ligand binding to protein is  Binding sites occupied
 MbO2 
Total binding sites
 MbO2    Mb
MbO
K A  Mb2O2 
K O 
O2 

 A 2 
K A  MbO2    Mb 
K A O2   1 O  1
 2 K
MbO  Mb


 2  
K A  MbO2 
K A O2 
O2 




K A  MbO2    Mb
K A O2   1 O  1
 2 K
Binding sites occupied


Total binding sites


A

O2 
O2   K D
A
O2 


O2   K D
A Hyperbola!!!!
θ=
Special Case: θ = .5 (or ½)
Po
O2 

1



O2   K D PO  P50 2
2
2
2 PO2  PO2  P50
2 PO2  PO2  P50
PO2  P50
Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
Quantitative description of Hemoglobin
binding to Oxygen
Quantitative description of Hemoglobin
binding to Oxygen


PO2


1 
P50
n


PO2

Log
 Log
1 
P50
n

Log
 n Log PO2  Log P50
1 
n = slope
Log P50 = intercept
Hill plot (Archibald Hill, 1910)
(Hyperbolic)
High oxygen affinity
Hemoglobin (sigmodial)
(linear)
Low oxygen affinity
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.
Hill constant (NH) is a measure of
cooperativity
NH = 1
No Cooperativity
NH > 1
Positive Cooperativity
NH < 1
Negative Cooperativity
Hemoglobin undergoes a structural
change when it binds to oxygen
Tense State (T-state)
Relaxed State (R-state)
Lysine 40
α-chain
Aspartate 93
β-chain
His 149
β-chain
Electrostatic interactions stabilize the
T-state of Hemoglobin
PDB ID: 1HGA
Asp
His
93
149
Lysine 40
α-chain
Lys
40
Lys
40
His
149
Asp
93
His 149
β-chain
Aspartate 93
β-chain
His 149
β1-chain
His 149
β2-chain
Electrostatic interactions stabilize the
T-state of Hemoglobin
PDB ID: 1BBB
Asp
His
93
149
His 149
β1-chain
Lys
40
Lys
40
His
149
Asp
93
His 149
β2-chain
Oxygen binding triggers a conformational
change from T-state to R-state
60 pm pucker
Valine
Leucine
Leucine
No Oxygen
T-state
No Oxygen
R-state
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).
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 Rstate.
• Hydrophobic interactions between the protein
and the of the porphyrin ring are stronger in the
R-state.
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
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
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
Concerted Model
Sequential Model
How does CO2 fit in?
(H+)-Hb
(H+)-Hb
Hb + H+
Hb + O2
Blood,
extracellular Fluid
Lungs, Air space
Effect of pH on the binding of oxygen
to Hemoglobin
Lungs
Tissues
Substitution of a Valine for a Glutamic acid on
the surface of Hemoglobin β-subunit is the cause
of Sickle-Cell Anemia
Normal Red Blood Cells
V-
Hydrophobic patch
Sickle-Cell Anemia Red Blood Cells
Protein Function II: The
Immune System
By
Doba Jackson, Ph.D.
Associate Professor of Chemistry and Biochemistry
Huntingdon College
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- Lymphocytes
T- helper cells (Th cells)
Major Histocompatability Complex (MHC)
• Cellular Immune System- uses receptors on the surface of TLymphocytes 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)
Important Lymphocytes
Lymphocytes 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 virusinfected or tumor cells and kill them.
The Complex Immune System
Cellular Immune System
Major Histocompatability Complexes
(MHC’s) is essential to the Cellular Immune
System
- Both MHCs have both an α and β chains
however, 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 βchain.
- 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.
The Complex Immune System
Helper T-cells activation
B-cells are activated using cell surface
antibodies and T-helper cells
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 MHC
proteins with the bound antigen. If the
bound antigen is foreign, the T-cell
receptor will lyse the cell and dispense
its contents.
Humoral Immune System uses
immunoglobulins (antibodies)
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.
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.
Different Immunoglobulin
subtypes occur in all B-cells
The Immune System is SelfTolerant
• 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 Bcells. These cells are destroyed upon birth.
• Occasionally, the immune system attacks its own
antigen after the selection period. This results in
autoimmune diseases.
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.
Induced fit in the binding of IgG to an
Antigen
The high affinity and specificity of antibodies make them
very useful for biological assays
ELISA
Enzyme-linked immunosorbant assay
Western Blot
Protein Function III: Muscle
Contraction
By
Doba Jackson, Ph.D.
Associate Professor of Chemistry and Biochemistry
Huntingdon College
Myosin has a globular amino
terminus and a long coiled coil tail
17 nm Head
Myosin, Actin Filaments
Striated Muscle Fibers
Back to step 1