Lecture 1-8

List of Biomolecules

Proteins

Nucleic Acids/Nucleotides

Carbohydrates

Lipids

Other(any molecule found in organisms and needed for biological processes)

Proteins

Rhino Horn(source of keratin), Red Blood Cells(source of hemoglobin), Firefly luciferase

Nucleic acids/nucleotides

DNA double helix, ATP

Carbohydrates

Sugar(sucrose), Cotton(cellulose), Insect shells(chitin)

Lipids

Waxes, Fat droplets in adipose tissue

Other Biomolecules

Metabolites, Vitamins, Antibiotics, Penicillin

hundreds or thousands 630 listed in one database(KEGG)

thousands in other databases(human, yeast, E. coli, metabolome databases)

What do biomolecules do?

Building blocks of cells and life

Fuel for Life

Engine for Life

Importance of Amino Acids

Building Blocks of proteins

What are amino acids named by?

have similar structure

named by R substituent(group)

The Five Groups based on R substituent(group)

Nonpolar, aliphatic R groups

Aromatic R groups

Polar, uncharged R groups

Positively charged R groups

Negatively charged R groups

Nonpolar, aliphatic R groups

nonpolar group makes them hydrophobic

aliphatic means they are hydrocarbons with straight or branches chains(not aromatic)

Glycine, Alamine, Proline, Valine, Leucine, Isoleucine, Methionine

members with simple structure R= -H, R= -CH3

Proline: cyclic structure that causes kinks in peptide chains

Methionine: contains sulfur

Aromatic R group

nonpolar, large aromatic groups

aromatic means they are cyclical(with bonds in resonance)

Phenylalanine, Tyrosine, Tryptophan

Tyrosine: Hydroxyl group forms hydrogen bonds and is important functional group in some enzymes

Polar, Uncharged R group

polar groups makes them hydrophilic

form hydrogen bonds

Serine, Threonine, Cysteine, Asparagine, Glutamine

Cysteine: contains sulfur and form disulfide bonds

Positively charged R groups

Positive charge makes them hydrophilic

Lysine, Arginine, Histidine

Histidine: serves as proton donor or acceptor in many enzymes

Negatively charged R groups

Negative charge makes them hydrophilic

Aspartate, Glutamate

Polar, Uncharged R group-Cysteine →disulfide bonds

Sulfur atoms of two cysteines can form a bond
Bond is important to protein structure and folding
Curls in hair are due to disulfide bonds
Straight hair can be made curly by chemical treatment

Titration curve

deprotonation of carboxylic group with increasing pH→shows pKa(pH where unprotonated and protonated forms are equal in concentration)
Same for amino group, but pH for deprotonation is higher

Carboxyl groups are ionizable

acidic pKa and is unprotonated at pH = 7

amino groups are ionizable

basic pKa and is protonated at pH = 7

R group can also be ionizable

Histidine has group with pKa = 6

-Near pH in cells (~ 7)
-Explains why it is a good proton donor or acceptor in enzymatic reactions

Positively charged R groups
◼ pKa >> 7 (except histidine)
Negatively charged R groups
◼ pKa << 7

Uncommon amino acids

many uncommon (nonstandard) amino acids
Usually not encoded directly by DNA
May or may not be part of protein

derived from common amino acids

Some are metabolites

example of Uncommon amino acids

4-hydroxyproline and hydroxylysine
◼ Formed by hydroxylation of proline and lysine
◼ Occurs after translation
◼ Part of collagen

phosphoserine and other phosphorylated amino acids
◼ Formed by phosphorylation
◼ Modification is reversible
◼ Done to regulate activity of proteins

ornithine and citrulline
◼ Formed during urea cycle (conversion of ammonia to urea)
◼ Not part of proteins

uncommon amino acid 21st and 22nd amino acids

Selenocysteine
Pyrrolysine
◼ Both encoded by DNA
◼ Both part of protein
◼ Not common

Amino acids polymerize to form peptides

Polymerization involves
 Carboxyl group of one amino acid
 Amino group another
Peptide bond is formed
An example of a condensation reaction

Peptide ends are not the same
 Numbering and naming startfrom the amino terminal end

Dipeptide-Two amino acids
Tripeptide-Three amino acids
Oligopeptide-Up to ~10 amino acids
Polypeptide-Up to ~100 amino acids (~10 kDa)
Protein-More than ~100 amino acids (>10 kDa)

Peptides form proteins

After polymerization, peptide folded into more complex structures

Primary-Amino Acid Residue

Secondary- alpha Helix

Tertiary-Polypeptide chain

Quaternary-Assembled subunit

Primary structure-simplest level

sequence of peptide
 Determined when amino acids polymerize

Secondary structure

short segments of peptide folded into regular arrangements
Two main types
◼ alpha helix
◼ beta sheet
Other types: Collagen helix(Found only in collagen), Random coil(No regular arrangement)

alpha helices are common secondary structures

Have a helical (corkscrew) shape: Held together by hydrogen bonds
◼ Oxygen of carboxyl group of one amino acid
◼ Hydrogen of amino group of second amino acid
◼ First amino acid is four residues away second amino acid
3.6 residues (5.4 Å) per turn
R substituents point out

Some amino acids are common or rare in alpha
helices

Common: Ala & Leu→ Have small, hydrophobic R groups, Pack well into helix

Rare: Proline
- N atom is part of rigid ring, and peptide bond cannot rotate
- Forms kinks in helix when present

Glycine
- Has tiny R group, giving it flexibility to make up other structures
- Tends to make up random coil, not helix

Beta sheets are another common secondary structure

Have pleated sheet-like structure
R groups (side chains) stick out from above and below the sheet

Beta Sheet secondary structure Top view

Made up of several strands
-Strands held together by hydrogen bonds between carboxyl group and amino groups

Parallel Beta sheet Top view

6.5 A

Antiparallel Beta Sheet Top view

7 A

Beta Turn

Part of Beta sheets
Occur whenever strands change the direction
Proline in position 2 or glycine in position 3 are common

Tertiary structures involve folded peptide chains

Involves entire peptide chain, not just short segments
Made up of alpha -helices, Beta sheets, or other
elements of secondary structure

Tertiary structures: 2 types

Fibrous
- Long strands or sheets
- Not soluble (in either water or lipid)
Globular
- Spherical shape
- Soluble (in either water or lipid)

Fibrous- tertiary structure

alpha-Keratin
- Found in hair
- Formed from coils of alpha-helix

Silk
- Formed by spiders and moths
- Made up of antiparallel beta sheets
- Small R groups (Ala and Gly) allow the close packing of sheets

Collagen
- Found in connective tissues
- Made up of collagen helices:
Different from -helix(e.g., 3 amino acid residues per turn)
Helices crosslinked together for strength
Crosslinks are covalent bonds between lysine, hydroxylysine, or histidine residues

α Helix, cross-linked by disulfide bonds

Tough, insoluble protective structures of varying hardness and flexibility

ex. α-Keratin of hair, feathers, nails

β Conformation

Soft, flexible filaments
ex. Silk fibroin

Collagen triple helix

High tensile strength, without stretch
ex. Collagen of tendons, bone matrix

Globular-tertiary structure

not as ordered as fibrous

ex. myoglobin

Often made up of motifs
- Contain two or more elements of secondary structure

• Beta-Alpha-Beta loop and Beta barrel

Often have multiple domains
- Single entities that are stable when separated from each other

Quaternary structure is the most complex

Formed by multiple peptides (elements of tertiary structure)

ex. Deoxyhemoglobin (has four peptide chains)

Protien’s folding

Folding is the process by which peptide chains develop tertiary or quaternary structure
- Done by chaperones (other proteins)
- Makes proteins functional

ex. Chaperones in E. coli: Serves as protective bubble for folding
- Protein enters and sealed inside
- Protein folds and is then released

How are proteins unfolded?

Proteins can be unfolded (denatured) by
- Heat
- pH extremes
- Organic solvents
-Chaotropic agents
◼ Disrupt hydrogen bonding
◼ Examples: urea and guanidinium hydrochloride

Ribonuclease refolding experiment

Tm= temperature at which half of protein is unfolded

Adding urea and 2-mercaptoethanol causes ribonuclease to unfold
Removing them allows it to spontaneously refold
Function restored when refolded

Misfolding is basis of many diseases

Creutzfeldt-Jakob disease
- One type of protein in brain (human prion protein) becomes misfolded
- One misfolded protein causes more to unfold, leading a domino effect
- Spongiform encephalopathy of brain and death result
Other diseases
- Bovine spongiform encephalopathy in cows
(mad cow disease)
- Scapie in sheep
- Chronic wasting disease in deer and elk

Methods to characterize proteins in the lab

SDS-PAGE-measure the size of proteins
Isoelectric focusing/2D PAGE→ can be done with SDS-PAGE
Chromatography/purification→ purifying large amounts of proteins
Mass spectrometry→ sequence of a proteins
X-ray diffraction→ crystallography reveals the structure

Gel electrophoresis separates proteins by size

Proteins loaded into wells of gel
Electric field applied
Proteins move (migrate) at speed according to size: Small proteins move through gel more easily

Gel is commonly polyacrylamide (or PAGE)
Proteins usually denatured by sodium dodecyl sulfate(SDS)-Technique called SDS-PAGE

Size (molecular weight) measured in comparison to standards

Isoelectric focusing separates proteins by charge

Proteins applied to gel strip
- Strip has pH gradient
Electric field applied
Proteins move until they reach pH= isoelectric point (pI)
- Isoelectric point is point at which proteins have no net charge

2D gel electrophoresis separates by size and charge

Isoelectric focusing done first (dimension 1)
SDS-PAGE done next (dimension 2)
Separation of complex mixtures possible

Western blot identifies proteins with antibodies

SDS-PAGE gel is run
Immunoblot is performed
- Proteins are transferred to a membrane
- Proteins in membrane are “probed” with antibody against protein of interest
- Dark color is formed, revealing protein(Series of reaction used to form color)

Chromatography is used to purify proteins

Useful for purifying (separating) large amounts of protein
- Protein loaded onto solid phase (porous matrix)
- Eluted from matrix using liquid phase
- Proteins with low affinity for solid phase migrate faster and come off first

Chromatography-Ion exchange

Ion exchange:
- Separates by charge
- Solid phase has affinity for positive ions (cation exchange)
- Or solid phase has affinity for negative ions (anion exchange)

Chromatography-Size exclusion

• Separates by size

• Solid phase has affinity for small molecules
  ◼Small molecules trapped in pores of phase

Chromatography-Affinity chromatography

Separates by binding ability
Solid phase has ligand
Ligand binds to (has affinity for) a target on protein
One target is histidine tag added by genetic modification

Several methods are combined to purify proteins

1. Crude cellular extract
2. Precipitation with ammonium sulfate
3. Ion-exchange chromatography
4. Size-exclusion chromatography
5. Affinity chromatography

Mass spectrometry reveals protein sequence

Peptide broken into fragments
Mass of fragments measured
Mass of different fragments reveals sequence

X-ray crystallography reveals 3D structure

Crystals of proteins are grown
Crystals put into X-ray beam
Pattern of X-ray diffraction reveals position of atoms (structure)

Protein functions

Structure (e.g., collagen)
Catalysis of chemical reactions (enzymes)
Transport of molecules (hemoglobin)
Defense against pathogens (antibodies)
Movement (actin, myosin)
Others

Enzymes bind to:

Antibodies bind to:

Collagen bind to:

all proteins bind to molecules

substrates

pathogens

collagen molecules

without binding, proteins cannot function

Proteins function by binding to ligands

he molecule bound is a ligand
Include ion, small molecule, macromolecules
Best studied example of ligand binding is oxygen binding to
hemoglobin and myoglobin

Hemoglobin binds oxygen as a ligand

Binds to and transports oxygen in blood
Four subunits

Oxygen is bound by heme
Iron (Fe2+) of heme is central
Fe2+ is site of O2 binding
Fe2+ also bound to myglobin (via His residue)

Myoglobin binds oxygen as a ligand

Stores oxygen in muscle cells
One subunit

Oxygen is bound by heme
Iron (Fe2+) of heme is central
Fe2+ is site of O2 binding
Fe2+ also bound to myglobin (via His residue)

Oxygen binding by myoglobin

The amount of oxygen bound increases with oxygen concentration
In myoglobin, relationship is simple

Binding Relationship between oxygen/ligands

Y = fraction of ligand sites occupied
[L] = concentration of ligand (M)
Kd = dissociation constant (M)

Dissociation constant (Kd)
Concentration of ligand where Y =0.5
The higher the value, the lower the binding (affinity)

Ligands and proteins have a wide range of dissociation constants

High affinity→ Biotin-Avidin

Antibody-antigen→10^-12-10^-8

Sequence-specific protein-DNA→ 10^-11-10^-7

Typical receptor-ligand interactions→10^-10-10^-5

Low affinity→ Enzyme-Substrate→ 10^-7-10^-3

Hemoglobin relationship is more complicated with oxygen

Sigmoid (S) shaped
Due to high and low affinity states(more later)

Allows high uptake of oxygen in lungs and release in tissues

Shape due to two different states (forms) of hemoglobin
Low affinity state(Present at low [O2])
High affinity state(Present at high [O2])

The protein morphs from the low to high affinity state by biding to more O2

cooperative binding

Happens in hemoglobin

Binding of one ligand causes more to bind
- When ligand binds to one subunit, remaining subunits are stabilized and bind more ligand

How does CO influence O2 with binding to ligands?

Carbon monoxide (CO) prevents O2 from binding to hemoglobin
- CO is in engine exhaust
- Explains why small amounts (ppm) are lethal

How are antibodies made and work?

Antibodies produced by immune system in response to foreign molecules
Binds target molecule tightly to
- Inactivates it
- Targets it for destruction
Billions of different antibodies can be produced

What do antibodies bind to?

Target molecule is antigen
Antigen is bound by antigen-binding sites

Induced Fit

Antibody changes shape (conformation) when binding to antigen
- Provides tight binding

Motor Proteins produce large movements in cell

Motor proteins bind to and move other molecules
Responsible for
- Muscle movement
- Crawling and swimming of cells
Example: myosin and actin in muscle

How is movement caused in muscles?

Muscle contracts when filaments of actin move towards each other
Movement is caused by myosin walking across and pulling actin

thin filament→ actin

thick filament→ myosin(motor proteins)

Power Stroke in Muscles

Walking in myosin is catalyzed by ATP
- Binding, hydrolysis, and release of ATP cause conformational changes

ATp binds to myosin head, causing dissociation from actin

As tightly bound ATP is hydrolyzed, a conformational change occurs. ADP and Pi remain associated with the myosin head

Myosin head attaches to actin filament, causing release of Pi

Pi release triggers a “power stroke”, a conformational change in the myosin head that moves actin and myosin filaments relative to one another, ADP is released in the process

Nucleotides and nucleic acids have many functions

Serve as enzymes (ribozymes)
Energy for metabolism (ATP)
Enzyme cofactors (NAD+)
Signal transduction (cAMP)

Gene expression (synthesis of proteins)

Nucleotides contain

Nitrogeneous base
Pentose
Phosphate

Bases are either purines or pyrimidines
Pentoses are either ribose (RNA) or 2’-deoxyribose (DNA)

Nucleoside

Nucleotide without phosphate
Names are different from respective nucleotides (and bases)

Nucleoside Examples

Adenine → base
Adenosine → nucleoside
Adenylate → nucleotide

Ribonucleotides

Components of RNA
Abbreviated as A, G, U, C

Deoxyribonucleotides

Components of DNA
Abbreviated as A, G, T, C
Can also be abbreviated as dA, dG, dT, dC to distinguish from ribonucleotides

Minor nucleotides in DNA

Methylated nucleotides
Example: 5-Methylcytodine- In eukaryotes, marks which genes should be inactive (epigenetic control)
In bacteria, protects DNA from host immune system (restriction enzymes)
Methyl group added after DNA synthesis

Minor nucleotides in RNA

Example: inosine
Found in some tRNAs (in “wobble position” of anticodon)
Made by deaminating adenosine

Polymerize by forming covalent bonds between nucleotides

Covalent bonds are phosphodiester linkages
Linkages create negatively charged backbone

DNA is a double helix- Who proposed the structure?

Structure proposed by James Watson and Francis Crick in 1953- H bonds between bases

A → T

C → G

Explained Chargraff’s rules and consistent with X-ray crystallography

Who showed that DNA was a double helix through X-ray crystallography?

Double helix structure previously shown by X-ray crystallography
Cross = helix
Shown by Rosalind Franklin and Maurice Wilkins

Helix information: major and minor grooves

Bases are 3.4 Å apart
There are 10.5 bases per turn (36 Å)

Two chains are complementary
They run antiparallel
- One runs 5’ to 3’(Numbers are with respect to ribose)
- Other runs opposite

What does the DNA helix suggest?

way for replication
- Strands separate
- New (daughter) strand synthesized(Using old (parent) strand as template), Done by DNA polymerase
- Two new molecules formed(Each with a parent and daughter strand)

Other forms of DNA

Watson-Crick structure is B form
A and Z forms also observed
- Only B and Z forms found in cells

RNA types: 2nd type of nucleic acid

Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
Ribozymes (RNA enzymes; includes rRNA)
Others
- Formed as a single strand/helix

Structure of RNA

When it a simple strand (with no internal base pairing), structure is a single helix

Single strand can form internal base pairs
- Leads to complex secondary structures: Hairpins, Loops, Bulges

Single strand can form internal base pairs: Leads to complex 3D structure