TOPICS IN (NANO) BIOTECHNOLOGY

Lecture 4

23rd October, 2006

PhD Course

mRNA lifetime

mRNA molecules are eventually degraded by the cell – by RNases

The lifetimes of different mRNAs vary considerably, and determine how much protein is made:

Lifetime of bacterial mRNA – about 3 minutes

Lifetime of eukaryotic mRNA varies from less than 30 minutes to more than 10 hours!

mRNA lifetime

The different lifetimes of mRNAs are controlled by nucleotide sequences in mRNA, most often in the 3’ untranslated region (the 3’ UTR)

Different lifetimes help control the level of each protein made from the different mRNAs

Different lifetimes a result of evolutionary fine-tuning---the stability of different mRNAs is tied to the needs of the cell

Prokaryotes vs Eukaryotes

Prokaryotes vs EukaryotesHow did prokaryotic v. eukaryotic mRNAs become so different in terms of introns?

While having introns has advantages, also has a cost---maintaining a larger genome and spending energy processing RNA transcripts

Many believe that prokaryotes once had introns also, but they were lost to allow reproducing more rapidly and efficiently (yeast now has few introns)

But others argue introns arose as parasitic mobile genetic elements, and present-day introns are the remains of these selfish elements

Translation• Translation describes the conversion of mRNA into protein

• Messenger RNA includes a sequence of nucleotides that corresponds with the sequence of amino acids in the protein, called the coding region

• This coding region is flanked by two regions that allow to know where to start and stop translating – the leader and the trailer

Translation

The ribosome

The ribosome

Translation

Translation

Translation

Translation

Translation_movie

The Genetic Code

The Genetic Code

The Genetic Code

Genetic Dictionary

• 61 Triplet codons that specify a specific amino acid, three codons are termination signals and do not code for an amino acid.

• Termination Codons = UAA, UAG, UGA

• Linear form using ribonucleotides that compose the letters in the mRNA molecules.

• Each code word contains 3 letters. Each triplet, called a codon, specifies one amino acid

Genetic Dictionary• The code is unambiguous (each triplet specifies one amino acid)

• The code is degenerate. Meaning that more than one triplet may code for a given amino acid which is the case in 18 of the 20 amino acids

• The code contains “start” and “stop” codons that initiate and stop translation

• The code is commaless. No internal “punctuation” exists. Codons are read one after another with no breaks

• The code is non-overlapping. Each ribonucleotide is a part of only ONE codon

• The code is “almost” universal. Few exceptions.

Protein synthesis

- proteins are the most diverse molecule (polymer) in existence- this diversity underlies their function

Type Function Examples

structural support

Insects and spiders use silk fibers to make their cocoons and webs. Collagen and elastin provide a fibrous network in connective tissue such as tendons and ligaments. Keratin is the protein of hair, horns, feathers and other appendages

storage storage of amino acidsOvalbumin is the protein of egg white used by the embryo. Casein is the major protein in milk. Plants synthesize vast quantities of storage proteins in their seeds

transport transport of building blocksHemoglobin transports oxygen throughout the body. Other proteins transport molecules across the cell membrane

hormonalcoordination of an organism’s activities

Insulin is a hormone secreted by the pancreas to help regulate the concentration of sugar in the blood

receptorresponse of a cell to chemical stimuli

Receptor built into the membrane of a nerve cell detect chemicals signals released by other nerve cells

contractile movementActin and myosin are responsible for the movement of muscles. Contractile proteins work in cilia and flagella to propel cells

defensive protection against disease Antibodies recognize bacteria and viruses

enzymaticacceleration of chemical reactions

Digestive enzymes hydrolyze the polymers in food

Proteins

Our life is maintained by molecular Our life is maintained by molecular network systemsnetwork systems

Molecular network system in a cell

Protein function

- proteins are the most diverse molecule (polymer) in existence and this diversity underlies their function

- diversity comes from the 20 different monomeric units (amino-acids) which comprise proteins

- the average protein in a bacterium is 150 ± 87 amino acids.

Diversity

Consider the number of combinations for a 100 amino acid protein

= 20100 <--- that’s a large number !

Definitions:amino-acid the monomer or basic building block peptide 2-20 amino acids (a small protein)

polypeptide/protein 20-2000 amino acids

Diversity

+H3N C COO–

R

H

Diversity in the chemical behavior of amino acids comes from the chemical nature of the R group

R = nonpolar (hydrophobic — carbon chains)R = polar (hydrophilic — substituents that can

hydrogen bond)R = charged group (acidic or basic)

The R group is commonly known as an amino acid sidechain

Amino acids

+H3N C COO–

H

H

amino group(pK1 ~ 9 )

acid group(pK2 ~ 3 )

Amino acids

Glycine(the simplest amino acid)

pI (isoelectric point) = pK1 + pK2 = (3 + 9)/2 = 6at pH 6, glycine is neutral

+H3N C COO–

R

H

glycine(the simplest amino acid)

R = H

Amino acids

- R group consists of carbon chains

leucine and isoleucine

are structural isomers

Non-polar amino acids

- R group consists of carbon chains

phenylalanine and tryptophan

have aromatic rings which are flat due to the double

bond network

Methionine has a sulphur atom in its side chain

proline has its R group bound to

the amino nitrogen to form a ring network

Non-polar amino acids

- R group consists of carbon, oxygen and nitrogen atoms - together they make the sidechain more hydrophilic

Asn and gln have an amide functional group

Ser and thr are a mix of carbon chains and hydroxyl functional groups (-OH). Cysteine has a thiol group (-SH) which is otherwise structurally similar

to serinebut not chemically similar

Polar amino acids

- R group has a charge at physiological pH (7.4). pK of the charged groups vary

carboxylgroup

carboxylgroup

aminogroup

guanidiniogroup

imidazolegroup

Polar amino acids

- amino acids have a full name (glycine), a short three-letter name (gly) and an even shorter one-letter name (G)

A ala alanineC cys cysteineD asp aspartic acidE glu glutamic acidF phe phenylalanineG gly glycineH his histidineI ile isoleucineK lys lysineL leu leucineM met methionineN asn asparagineP pro prolineQ gln glutamineR arg arginineS ser serineT thr threonineV val valineW trp tryptophanY tyr tyrosine

nonpolar

polar

acidic (negative charge)

basic (positive charge)

Describing amino acids

L- vs D- configuration

The Peptide Bond

- in a cell, a complex assembly of proteins and RNA called a ribosome catalyse a dehydration reaction (loss of water) to join amino acids together

loss of water

The ribosome does not join an

amino acid to this end

(the amino end)

The ribosome only joins new amino acids to this end(the carboxy end)

chain extends in

this direction

Joining amino acids

- a peptide bond (like an amide bond C-O-N) joins each amino acid- the invariant purple part of the polypeptide is generally called the backbone- it’s the sidechains that give a protein its unique chemical character

Joining amino acids

Protein architecture

- lysozyme is a protein found in egg white that has anti-bacterial properties. It is an enzyme which catalyses the breakdown of a polysaccharide network necessary to maintain the integrity of the bacterium.

- there are 129 amino acids (or residues) in lysozyme. The amino and carboxyl ends are free (not bound to anything else)

- the sequence of amino acids is called the primary structure

Primary Structure

- the protein spontaneously folds to minimize hydrophobic (nonpolar) sidechain exposure to water and maximize hydrophilic (polar and charged) sidechain exposure to water.

- the HN (amide) and CO (carbonyl) groups of the backbones have covalent bonds which are polarized much like water

- the protein also folds up to encourage a hydrogen bond between the the HN and CO groups

Much farther than 2.4 Å so the protein folds up to make H-bonds

N–H ------ O=C

hydrogen bond1.8 to 2.4 Å in length

Secondary Structure

- the alpha helix (a-helix) is one common form of secondary structure

- much like the coils of a telephone cable

- due to the hydrogen bonding network in an alpha helix, this structure is stable

residue n

residue n+4

residue n+8

Secondary Structure

- the beta sheet (-sheet) is another common form of secondary structure much like the pleats of an accordion

- beta sheets can join very distant parts of the protein together

- due to the hydrogen bonding network, beta sheets are very stable

Secondary Structure

- the protein spontaneously folds to minimize hydrophobic (nonpolar) sidechain exposure to water and maximize hydrophilic (polar and charged) sidechain exposure to water.

-helix -sheet

extended

loop

Tertiary Structure

- the active configuration of protein may consist of more than one folded protein unit

- three collagen chains twist into a strong fiber

Quaternary Structure

- the active configuration of protein may consist of more than one folded protein unit

- three collagen chains twist into a strong fiber

- two alpha subunits and two beta subunits combine to form a functional molecule of hemoglobin. Each subunit bind one molecule of heme, an iron containing cofactor which helps bind oxygen

Quaternary Structure

- in addition to hydrogen bonds and the force to minimize the exposure of hydrophobic amino acid sidechains, there are other mechanisms that assist folding

- disulfide bonds occur between two cysteines

- a positively charged sidechain may form an ionic bond with a negatively charge sidechain (lysine -> aspartate)

Protein folding

- temperature (heat), pH and solvent conditions can be adjusted to unfold a protein back into a more extended form.

- when the unfolding conditions are reverted, many proteins have enough information stored in their sequence of amino acids to refold back to exactly the same tertiary structure. Other proteins get stuck along the way (curdled milk stays curdled after heat/cool treatment)

-much research is done to solve the protein folding problem, or given a sequence, can one predict how the protein will fold up.

-http://www.sumanasinc.com/webcontent/anisamples/nonmajorsbiology/proteinstructure.html

Protein folding

Hierarchical nature of protein Hierarchical nature of protein structurestructure

Primary structure (Amino acid sequence)↓

Secondary structure （ -helix, β-sheet ）↓

Tertiary structure （ Three-dimensional structure formed by assembly of secondary structures ）

↓Quaternary structure （ Structure formed by more than

one polypeptide chains ）

α-helix β-sheet

Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.

Protein architecture

Three-dimensional structure of Three-dimensional structure of proteinsproteins

Tertiary structure

Quaternary structure

Mutatation• Mutations change the sequence of DNA

• Mutations can be spontaneous or induced

- sickle cell anemia is caused by a point mutation in hemoglobin b chain (a is unaffected)

val-his-leu-thr-pro-glu-glu … normal individualval-his-leu-thr-pro-val-glu … affected individual

- only one amino acid is changed in the entire sequence of the protein

glutamic acid sidechain -CH2-CH2-COO– acidic sidechainvaline sidechain -CH-(CH3)2 nonpolar sidechain

- the hemoglobin molecule folds up and functions (binds oxygen) but the mutation caused the protein to clump up in the cells. The clumping up distorts the cell shape and makes them architecturally weaker.

Sickle Cell Anaemia

- the surface of the protein has sidechains sticking out. Polar and charged sidechains help the protein stay dissolved in water

- the glutamic acid to valine mutation is a surface mutation

Sickle Cell Anaemia

- mutations are responsible for numerous diseases

- cystic fibrosis (point mutation)- Huntington’s disease (insertion of extra amino acids)

- HIV uses mutations to its advantage

- a drug that binds to an HIV protein may not bind very well only a few viral generations later

- structural biologists study the relationship between protein structure and protein function

- to design new or better drugs- to understand how proteins are constructed- (nature tends to use the same motif over and over

again)

Mutations

- a single polypeptide chain often consists of a number of smaller autonomously folding units called domains. Sometimes they arranged like beads on a string…

H3N COOHactivity 1 activity 2 activity 3

- often though, each domain interacts with the others- much like quaternary structure built into ternary structure- over evolutionary time, the genes that encode each module/domain get shuffled and spliced to make new proteins

activity 1

activity 2activity 3

Modular nature of proteins

An important set of proteins: Enzymes

• Thousands of biochemical reactions proceed at any given instant within living cells. These reactions are catalyzed by enzymes;

• Enzymes are mostly proteins. But two important enzymes are most certainly to be RNA (ribozymes). One is the ribosome (peptidyl transfer) and the other is the spliceosome (splicing of intron);

• Enzymes are the agents of metabolic function. Enzymes play key functions in controlling rate of reaction, coupling reactions, and sensing the momentary metabolic needs of the cell.

Enzymes

Enzymatic Catalysis Suited to Biological systems

• Higher reactions rates• Milder reaction conditions• Greater reaction specificity• Capacity for regulation

Enzyme-substrate interactions-a prerequisite for catalysis

• Forces Important for substrate recognition

• Active Site Characteristics

LOCK AND KEY INDUCED FIT

Models for Enzyme Substrate Interactions

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O

O

P

O

O

O

P

O

O

O

-

- - -

ATP

Enzyme Cofactors

+

N

H

2 O

OO

O CH

H H

H

P

O

O-

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O-

O

O

C-NH2

H

+N

H

2 O

OO

O CH

H H

H

P

O

O-

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O-

O

O

C-NH2

H H

+ H + 2e+ -

-- H - 2e

..

NAD NADH+

Enzyme Cofactors

HS-CH -CH -N-C-CH -CH -N-C-C-C-CH-

N

N

N

H

2 O

OO

ON

N

CH

H2

H H

H

P

O

O-

OP

O

O22

O

H

CoASH

O

-

2

O OH

H

CH3

CH3

2H

2

Enzyme Cofactors

23 3

ALCOHOL DEHYDROGENASE

CH -CH -OH + NAD CH -CH=O + NADH + H+ +

Enzyme Classifications

Oxido-reductases

Transferases

Enzyme Classifications

Hydrolases

PROTEASE

R-NH -CH-C-NH-CH-C-NH-R

OO

R R1 2

+ H O2

R-NH-CH-C-OH

R1

O

NH -CH-C-NH-R2

R2

O

+

Lyases

ENOLASE

O

O

O P-

-

CH -OH

O-C-H

C-O

O

- O

O

O P-

- C-O

O

2

-

CH

O-C + H O2

2

Enzyme Classifications

Isomerases

Ligases

CO H

C

H N

2

CH3H

CO H

CHN

2

CH3H

D-ALANINE

L-ALANINE

3

3

+

+

CO H

C

2

CH3

O + NH +

4

D-AMINO ACID OXIDASE

Enzymatic Reactions with Stereochemical Specificity

23 3

ALCOHOL DEHYDROGENASE

CH -CH -OH + NAD CH -CH=O + NADH + H+ +

An important set of proteins: Antibodies

So what is an antibody?

• Antibody

So, what is an antibody?

What is an antigen?

Any substance capable of producing a specific immune

response

So, what is an antibody?

B cells and T cellsThe two major classes of lymphocytes are B cells, which grow to maturity in the bone marrow, and T cells, which mature in the thymus, high in the chest behind the breastbone.

B cells produce antibodies that circulate in the blood and lymph streams and attach to foreign antigens to mark them for destruction by other immune cells.

B cells are part of what is known as antibody-mediated or humoral immunity, so called because the antibodies circulate in blood and lymph, which the ancient Greeks called, the body's "humors."

B cells and T cells

B cells become plasma cells, which produce antibodies when a foreign antigen triggers the immune response.

B cells and T cells

Certain T cells, which also patrol the blood and lymph for foreign invaders, can do more than mark the antigens; they attack and destroy diseased cells they recognize as foreign.

T lymphocytes are responsible for cell-mediated immunity (or cellular immunity).

T cells also orchestrate, regulate and coordinate the overall immune response.

T cells depend on unique cell surface molecules called the major histocompatibility complex (MHC) to help them recognize antigen fragments.

Recognition of antigen by B and T-cells

• B-cells can recognise an epitope alone• T-cells can recognise antigen only when

it is associated with an MHC molecule• There are four cell membrane molecules

that are involved in recognition:– membrane bound antibody (B-cells)– T-cell receptor or TCR (T-cells)– MHC class I– MHC class II

What is an antibody?

• Antigen-specific products of B-cells• Present on the B-cell surface • Secreted by plasma cells• Effectors of the humoral immune response,

searching and neutralising/eliminate antigens• Two functions:

– to bind specifically to molecules from the pathogen

– to recruit other cells and molecules to destroy the pathogen once the antibody is bound to it

Structure of the antibody molecule

• The antigen-binding region of the antibody molecule is called the variable region or V region

• The region of the antibody molecule that engages the effector functions of the immune system is known as the constant region or C region.

• They are joined by a polypeptide chain that is known as the hinge region

Structure of the antibody molecule

• X-ray crystallography has revealed that the overall shape is roughly that of a Y

• Each arm of the Y is formed by the association of a light chain with a heavy chain

• The leg of the Y is formed by the pairing of the carboxyl-terminal halves of two heavy chains

Light Chain

• There are two types of light chain– kappa (k) chains– lambda (l) chains

• No functional difference has been found between antibodies having l or k light chains

• In humans 60% of the light chains are k, and 40% are l

Heavy chain

• There are five heavy chain classes or isotypes– IgM (m chain)– IgD (d chain)– IgG (g chain)– IgA (a chain)– IgE (e chain)

• These determine the functional activity of an antibody molecule

IgG

• IgG– most abundant

immunoglobulin in the blood

– provides the bulk of immunity to most blood-borne infections

IgD

• IgD– present in low

quantities in circulation

– primary function is that of antigen receptor on B-cells

IgE

• IgE– present in the serum

at very low levels– plays a role in acute

inflammation and infection by parasites

IgA

• IgA– present in external

secretions, such as tears, milk, saliva

– first line of defense against microbial invaders at mucosal surfaces

IgM

• IgM– first antibody produced

and expressed on the surface of B-cells, also secreted

– 10 combining binding sites per molecule make it very effective in removal of microbes

Enzyme Linked ImmunoSorbent

Assay (ELISA)

ELISA

• An analytical method based on the exploitation of the highly specific and selective nature of antibodies

• Radioimmunoassay developed in mid-sixties and the first report of enzyme immunoassay was in 1976 (Rubenstein et al.)

How do we produce polyclonal and monoclonal antibodies?

Polyclonal antibodies

- larger quantities may be produced at a time

- sometimes better selectivity and sensitivity due to recogintion of multiple epitopes

- no guarantee of batch to batch reproducibility

Monoclonal antibodies

- long and expensive process

- sometimes lower selectivity and sensitivity in comparison to Pabs observed

- once cell line established constant reproducible supply of antibodies …. forever

Enzyme Labels• Enzymes are protein catalysts present in all living cells.• They catalyse all essential reactions to supply the energy and/or

chemical chnages necessary for vital activities.• Enzymes bind their corresponding substrates with high specificity.

E + S ES E + P• Release of this product may be monitored by measuring, for

example, colour change.

• With the substrate in excess, the signal observed is proportional to the amount of enzyme present.

• Following enzymatic action, the products of the reaction are released and the enzyme is free to bind another substrate molecule.

• The speed with which this occurs is known as the turnover rate.

• Enzymes are conjugated to antibodies to provide a means of measuring the mount of antibody present.

• Enzymes commonly used are horse radish peroxidase, alkaline phosphatase, -galactosidase and glucose oxidase.

ENZYME SUBSTRATE (nm)

Horseradish peroxidase o-phenylenediamine dihydrochloride (OPD) 492*

tetramethylbenzidine (TMB) 450*

2,2’-azino-di-(3-ethyl)benzthiazoline 414* sulphonic acid (ABTS)

5-aminosalicyclic acid (ASA) 450*

[* H2O2 added and reaction stopped with sulphuric acid]

Alkaline phosphatase p-nitrophenyl phosphate 405

-D-galactosidase o-nitrophenyl -D-galactosidase 405

Glucose oxidase Glucose

(H2O2 produced and HRP and substrate used)

HRP

TMB/OPD/APTS

(no colour)

Oxidised product

ALP

p-nitrophenylphosphate

(no colour)

p-nitrophenol

-GAL

p-nitrophenylgalacto-pyronasidase

(no colour)

p-nitrophenol

Measurement principle

Note: Can also label antigen with enzyme!

Microtitre plates

96-well ELISA plate

Surface of polystyrene is activated with amine groups for enhanced binding of antibody

NUNC plates - best well to well reproducibility in binding (also very useful web site www.nunc.com)

With the exception of checkerboard titrations, avoid using column 1 and 12 and rows A and H, due to uneven heating effects

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Sandwich assay

substrate

product

substrate

product

substrate

product

Concentration

Res

po

ns

e

Useful for large molecules

Robust assay - all reagents in excess

Use with Pabs or different MAbs

Competition assay

substrat

e

product

substrat

e

product

Concentration

Res

po

nse

Useful for small molecules

Reportedly less sensitive

Concentrations of reagents critical

Displacement assay

substrat

e

product

substrat

e

product

Concentration

Res

po

nse One step assay

In practise difficulties to achieve - effect of non specific displacement

Sub-optimum haptens met with some success

Aptamers are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment via an in vitro iterative process of adsorption, recovery and reamplification, known as SELEX (systematic evolution of ligands by exponential enrichment).

APTAMER DEFINITIONAPTAMER DEFINITION

Artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and even cells.

They bind their target with selectivity, specificity and affinity equal and often superior to those of antibodies.

SELEXSELEX

SELEX

can be selected against toxins/molecules that do not elicit good immune response selection is in vitro process - does not need animals kinetic parameters (kon/koff) can be controlled can be regenerated in minutes, stable for long term storage, can be transported at ambient temperature can be used in non-physiological conditions produced by chemical synthesis no ‘batch to batch’ variation negative selection against structures similar to target structure can improve specificity

BUT low stability = short life

Can be solved by chemical modification, spiegelmers, mixed LNA/DNA structures

APTAMERS VS. ANTIBODIES

Aptamers vs Antibodies

Examples of molecules for which aptamers have been selected in vitro:

ATPArginine

Dopamine Reverse transcriptase of HIV

ThrombineMembrane receptors

Whole viruses

Structure of aptamers

Structure

Modes of assay

Molecular beacons

• Molecular beacons essentially contain two structural components, a loop and a stem, with the loop serving as a probe and the annealing of two complementary arm sequences that are flanked by the probe forms the stem.

• A fluorophore and fluorescent quencher are linked covalently at each end of the arm. The stem of the beacon brings the fluorophore and quencher into close proximity, resulting in no fluorescent signal.

Molecular beacons

• When the molecular beacon encounters a target molecule it forms a probe target hybrid that is stronger and more stable than the stem in the hairpin, with the resulting conformational change forcing the arms apart, thus permitting the fluorophore to fluoresce.

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules.

FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity.

Primary Conditions for FRETDonor and acceptor molecules must be in close proximity (typically 10–100 Å).

The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor (see figure).

Donor and acceptor transition dipole orientations must be approximately parallel.

Fluorescence Resonance Energy Transfer (FRET)