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

Back to step 1