Lesson 1:Proteins

ProteinsMade of amino acids linked by peptide bondsThere are 20 types of amino acids that we use (though many more exist)

Be able to draw a generalized amino acid!

The Amino Acids

Condensation and Hydrolysis

Condensation: joins amino acids into a dipeptide or polypeptide, produces water

Hydrolysis: breaks a polypeptide into amino acids, uses water

Peptide bondsAlthough “peptide bond” refers specifically to the bond C-N bond formed between two amino acids, it refers to a larger structure:

The dark grey bond is the peptide bond, but the entire pink area is needed .

Be able to draw two amino acids forming a peptide bond!

Primary structure Number (can be ~50–1000) and order of amino acids in a chain (polypeptide) held together by peptide bonds

Each polypeptide is coded for by a gene (in the DNA).

Enormous range of possibilities! (20n, n=# of amino acids)

Type / location of amino acids used relate to the protein’s function

Secondary structureHeld together by hydrogen bondsRegular and repeating, formed by N-C-C backboneTwo main forms

-helix (myosin, hair) -pleated sheet (silk)

Tertiary structure Final 3-dimensional folded shape of polypeptide R-groups mostly determine bonding; may include:

ionic bondsHydrogen bondscovalent (disulfide) bondshydrophobic interactions

Quaternary structure(Only in some proteins) 2+ polypeptide chains held together by all types of bonds, primarily through R-groupsConjugated proteins also include elements and structures called prosthetic groups that are not amino acids (part of quaternary structure)

Protein StructureReview

Primary - order of amino acidsSecondary - H-bonds in backboneTertiary - 3D shape from R-groupsSOMETIMES Quaternary - 2+ polypeptides and prosthetic groups

What levels can you find?

Explore the biotopics jsmol library. You should compare and contrast: glucagon, myogloblin, and hemoglobin.

Functions of proteinsStructure and support - microtubules influence cell shape, collagen in skin, spider silk in websTransport - hemoglobin carries oxygen, microtubules serve as highways in cells Movement - actin and myosin make muscle fibersCommunication - hormones, ex. insulinDefense – immunoglobin antibodies, lysozyme in tearsResponse – response, eg. rhodopsin in eyesEnzymatic reactions - speed up reactions, eg. Digestion of food, building cell parts, Rubisco in photosynthesisEnergy storage - ~4 Cal/gram (not considered a primary function)

Final Protein shapes: Fibrous

Fibrous - long, thin, insoluble in water

Collagen: STRUCTURE in skin, tendonsActin and myosin: muscle fibers allow MOVEMENT

Final protein shapes: Globular

Globular - rounded, bulky, mostly soluble in water

Hemoglobin: TRANSPORT oxygen in red blood cellsImmunoglobin: antibody DEFENSE against foreign substances

Significance of amino acid variety

The different R-groups allow a variety of shapes

The active sites of enzymes have the correct polarity and/or charge to attract the substratesNon-polar amino acids can be anchored in non-polar membranesA membrane channel protein can have non-polar R-groups on the outside and polar R-groups on the inside, creating a hydrophilic passageway through the membrane

Final Protein Shape Depends on Environment

A protein that has lost its function (usually permanent) is “denatured”Excessive heat (not cold) or unsuitable pH (acidic or alkaline) can disrupt bonding and denature the protein

Look at the production of “century eggs” (ไข่�เยี่��ยี่วม้า) – using pH to denature!

Genes and ProteinsDNA is for information storage only, while proteins have a huge variety of functions.Each individual of a species has a slightly different set of DNA (genome).Each gene in the DNA codes for a protein

Almost all species have the same code for translating from DNA to protein sequence!

Each individual has a unique proteome, influenced primarily by DNA but also by environment (stimuli like stress can turn genes on or off).

Discuss the connections between stem cell differentiation and proteomes.

Map of C. elegans (nematode worm) proteins

and their interactions

Lesson 2:Enzymes

Enzymes Made of protein

Globular Catalyze ONE (or very few) specific

reactions Speed up rate of reaction Lower the activation energy of a reaction Very specific to substrate molecule(s)

Activation Energy (Ea)

Collision TheoryIn liquids (like cytoplasm) molecules are moving around, knocking into each other (colliding)When some molecules strike at the right orientation, with enough energy they will react (split apart, combine, shift bonds, etc)

The rate of reaction without an enzyme can be noticeable, or it can be so rare that it’s basically never.

Enzyme structure The active site is where

the substrate binds (other molecules bounce off)

The enzyme is like a lock, and the substrate(s) are like the key(s) that fits it

Enzymes and Collision Theory

Enzymes capture substrate(s) that collide with the active site and hold them at an angle that places stress on existing bonds and aligns substrates (if more than one)This increases the rate of reaction, often by millions of times.

Which factors affect enzyme activity?

[ ] means concentration

Temperature = increased energy increased collisions increased rate, BUT increased too much will break bonds, denature the structure

pH = [H+] affects bonding, attracting or repulsing R-groups, can denature the protein

[Substrate] = at low levels more substrate increases rate of reaction because more collision, at high levels no effect because enzymes already saturated

Rate of reaction is also affected by [Enzyme] = a higher concentration will lead to more collisions, therefore more reactions

Factors Affecting Enzyme Function

Temperature

pH

[Substrate]

[Enzyme]

Be able to draw these graphs and discuss what is happening at each part!

Enzymes are evolved to work in their environments

Eg. All enzymes can be denatured by a pH that is unsuitable, but which pH is optimal (best) depends on the enzyme

Using animations to collect data

We will use this animation for modeling enzyme activity.

Inhibition of EnzymesCompetitive Substrate and inhibitor

both bind to active site Inhibitor and substrate

are often chemically related

Inhibitor physically blocks substrate

Non-competitive Substrate binds to

active site, inhibitor binds to allosteric site

Inhibitor and substrate not chemically similar

Inhibitor changes the shape of the enzyme and active site

Inhibition of Enzymes, cont.

Competitive Non-competitiveIn which case would the [substrate]

affect the rate of reaction?

Answer: Competitive; more substrate molecules will more successfully for the active site against the inhibitor

Competitive inhibition

• Ethanol is the alcohol found in drinks• Ethanol is converted to acetaldehyde• Aldehyde dehydrogenase immediately converts acetaldehyde to acetate so it never builds up•Further enzymes modify acetate for energy release or energy storage

• Disulfiram binds to the active site of aldehyde dehydrogenase, so acetaldehyde builds up causing nausea and discomfort• Used as a pill to treat alcoholism

Non-competitive inhibition Lead replaces zinc at an

allosteric site in aminolevulinic acid dehydratase (ALAD), an enzyme that helps produce hemoglobin

Changes shape of active site so ALAD does not function, leading to anemia

Lead inhibits many enzymes leading to many other symptoms including headache, insomnia, insanity, death

Why do both types of inhibition have an increased rate of reaction at low levels of [substrate]? Why does non-competitive inhibition show no effect from [substrate] at higher levels?

Compare the three conditions.

Metabolic pathways Many reactions are actually a

series of steps, each catalyzed by a different enzyme, in a chain or cycle

End-product inhibition -- Negative feedback

Helps maintain balance (homeostasis) by preventing overproduction

The final product serves as an allosteric inhibitor for an enzyme early in the pathway

End-product inhibition in threonine isoleucine

pathwayIsoleucine is an allosteric inhibitor of threonine deaminase, the first enzyme in the metabolic pathway that converts threonine to isoleucine.

Discuss the role(s) of:•Threonine•Threonine deaminase•Isoleucine

What happens when levels of isoleucine are low? High?

Use of lactase enzyme in lactose-free milk production Specific yeast is cultured to

harvest its lactase Lactase breaks lactose

disaccharide into glucose and galactose

People who lack this enzyme are lactose-intolerant

May have diarrhea, gas, and intestinal pain when eating dairy products

Lactase can be added to produce lactose-free dairy foods

Lactose Free Milk

MethodsAdd lactase directly to

milkImmobilize lactase on

a screen and slowly pour milk over (no lactase in final product)Non-enzyme method

Ultrafiltration

AdvantagesSweeter taste Digestible by

lactose-intolerant people

Fewer allergiesIf made into ice

cream, less grittyIf made into yogurt,

process is faster

Uses of immobilized enzymes

Removal of wastes from contaminated water

Pectinase and cellulase to release juice

Antibiotic production

Much more!

Using Databases to look for Anti-Malarial Drug

TargetsDeveloping medicines is extremely expensive and time consumingDatabases and pool knowledge and lab resultsAllows selection of most useful enzymes to target, or drugs that are known to be tolerated in humansEfficient and more rapid development

Read this abstract. What did the authors do?Explore the research database site TDRtargets.org. What makes a gene a good target?Links allow further exploration, including amino acid sequencing (see BRENDA)

Optional Challenge Activity!

Are you a gamer?Want a real challenge?Want to make a difference?Try Foldit!

Learn how amino acids interactLike a crazy-hard puzzle

Read an article here: Gamers took 3 weeks to solve a protein researchers had worked on for 10 years!

Can be used by HS students – see how far you can go!!

Lesson 3:Nucleic Acids

Nucleic Acid StructureNucleotide:Sugar (ribose /

deoxyribose)

PhosphateNitrogen Base

Nucleic acids are chains of covalently-bonded nucleotides

Nucleotides are the monomers (building blocks) of nucleic acids

Each nucleotide has thee parts.

Nucleic Acid FunctionsNucleic acids polymers include:

DNA: stores genetic informationRNA: relays relevant information from DNA to the rest of the cell; directs the production of proteins

One nucleotide (monomer):ATP: the energy currency used in cells

The Nitrogenous Bases Purines – two rings, the same in DNA and RNA

AdenineGuanine

Pyrimidines – one ringCytosineThymine (in DNA)Uracil (in RNA)

Nucleotides form Nucleic

AcidsEnergy is released from nucleoside triphosphates; two phosphates break away.

The phosphate of one nucleotide links to the 3rd carbon in the sugar of another

Creates a covalently bonded phosphate – sugar backbone

The DNA Double HelixNitrogen bases form specific (complementary) pairs

Adenine pairs with Thymine (A – T)Guanine pairs with Cytosine (G – C)

Notice that a purine always pairs with a pyrimidineEach complementary base pair forms hydrogen bonds

A-T form 2 hydrogen bondsG-C form 3 hydrogen bonds

DNA Double helix•Antiparallel

strands: in order for the nitrogen bases to form hydrogen bonds, the two DNA strands must be facing opposite directions

•Constant width: Because a small pyrimidine always bonds with a larger purine, the two strands are always the same distance apart

DNA double helix•The sugar-phosphate

backbones are on the outside of the helix

•The nitrogen bases form the flat inner rungs (steps on the ladder) of DNA

•If you know the order of Nitrogen bases on one strand of DNA, you can determine the other:

•Practice: ACTTGCCA

•Answer: TGAACGGT

DNA packaging in Eukaryotes

•In eukaryotes and archaea (NOT eubacteria) DNA is organized into nucleosomes:– 8 histone proteins, 2 loops of DNA, one histone

“tie”

DNA to

Chromosomes• DNA must be

uncoiled for the information to be “read” (transcribed)• DNA must be supercoiled when not in use or it will tangle and tear •Histone proteins organize the DNA by winding it up

RNA types•mRNA (messenger)

– Carries a copy information from individual genes to ribosomes (as needed)

•rRNA (ribosomal)– Acts as a catalyst (enzyme,

except not protein) joining amino acids into a polypeptide.

•tRNA (transfer)– Translates from nucleotide code

to assemble correct amino acid sequence

RNA v. DNA

Nucleic acid polym

er

# of strand

s

Nitrogenous bases

Pentose sugar Function

DNA 2, double helix

Thymine, Adenine, Guanine,Cytosine

Deoxyribose Genetics, information storage

RNA 1, not a helix

Uracil, Adenine, Guanine,Cytosine

Ribose Information transfer (DNA to protein), protein synthesis

Hershey-Chase Experiment, 1952

• For a long time, protein was considered most likely to be the genetic material– Complex enough to

store large amounts of information

• In the 1940s, evidence started to accumulate that DNA might be the genetic material– Led to many

researchers racing to determine the structure!

Hershey-Chase showed that that bacteriophage viral proteins do not infect bacteria cells, but viral DNA does!

Hershey Chase – experimental procedure

The Structure of DNA:The Great Race!

Three teams:

Caltech Cambridge King’s College

Linus Pauling James Watson & Francis Crick

Rosalind Franklin / Maurice Wilkins

Franklin (and Wilkins): X-ray crystallography

Photo 51: Go through this interactive.

Know at least that:•The “X” suggests a helix•The 4 white “diamonds” suggest a repeating helix•The “missing band” in the X suggests a double helix

Watson and Crick models• Like an early type of

“foldit” using cardboard cutouts of the different pieces

• Made to accurately represent bond length and atomic location

• Relies on modeler’s understanding of how elements would interact

• Published paper 1953