3-d structure of proteins by doba jackson, ph.d
TRANSCRIPT
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
• 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
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? S G Y A L
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
• 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
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
Repeated structure of Gly-X-Proline
Triple helix of Collagen
Structure of Collagen
Right-Handed or Left-Handed?
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
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 Mesh Diagram Mesh Diagram
Ribbon Diagram /w side chains
Surface DiagramUsing van der Waals radii
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 βαβ-motif as a fundamental unit
Triose Phosphate Isomerase
Quaternary Structure
Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8Ǻ
Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8Ǻ
Nonpolar residues
Structure of Horse Heart Cytochrome CPDB ID: 3CYT
Res: 1.8ǺCharged polar residues
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 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
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?
Hemoglobin: Protein Function in a Microcosm
By Doba Jackson
Assistant Professor of Chemistry & BiochemistryHuntingdon College
Iron-Porphyrin complex (Heme)
Pyrole Pyrole
Pyrole Pyrole
Proprionate
Vinyl
Vinyl
Methyl Methyl
Methyl
Methyl
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
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
Distal His coordinates To the second oxygen
Proximal His
ProteinOxygenNitrogenCarbon
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
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
Structure of Hemoglobin demonstrates symmetry in its quaternary structure
α Subunit
α Subunit
β Subunit
β Subunit
Two-fold axis
Two-fold axis
Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
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
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
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
Myoglobin (Hyperbolic)
High oxygen affinity
Hemoglobin (Sigmodial)
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
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
Hill plot (Archibald Hill, 1910)
(linear)Low oxygen affinity
(Hyperbolic) High oxygen affinity
Hemoglobin (sigmodial)
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 40α-chain
His 149β-chain
Aspartate 93β-chain
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
His 149β1-chain
His 149β2-chain
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
Oxygen binding triggers a conformational change from T-state to R-state
No OxygenT-state
No OxygenR-state
60 pm puckerValine
Leucine
Leucine
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 R-state.
• 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
Blood, extracellular Fluid
Lungs, Air space
How does CO2 fit in?
(H+)-Hb Hb + H+
(H+)-Hb Hb + O2
Effect of pH on the binding of oxygen to Hemoglobin
Lungs
Tissues
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
Protein Function II: The Immune System
By Doba Jackson, Ph.D.
Associate Professor of Chemistry and BiochemistryHuntingdon 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- 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)
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.
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 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.
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 MHCproteins 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 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.
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 BiochemistryHuntingdon College
Myosin has a globular amino terminus and a long coiled coil tail
17 nm Head
Myosin, Actin Filaments
Striated Muscle Fibers
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