Biol 200 Midterm 1

What defines living matter?

1. A high degree of chemical and

structural complexity

2. A capacity to extract, transform and

use energy from the environment

3. Defined functions for the organism’s

components and regulated

interactions among them

4. A capacity to sense and respond to

environment

5. A capacity to self-replicate

6. A capacity to evolve

What does life need to function? And how does it remain functional?

• complex macromolecules

• continuous input of energy

Lipid Barrier (cell’s plasma membrane)

• serves as a selective barrier, allows greater

concentrations of nutrients and synthesized products inside the cells than outside

• Allows cells to have a distinct

intracellular environment separate

from its surroundings (to be outside of a

chemical equilibrium with its

surroundings)

Proteins

• each have a specific function

• very versatile in what functions it can serve

  • structural roles, generate movement, sense and

    detect signals, catalyzing reactions (including those needed to obtain and use energy from the

    environment)

    
    

    
    

DNA

• contains information to self-replicate

• simpler than proteins

  • only four building blocks vs 20 amino acids for proteins

Central Dogma

The flow of information from DNA to the production of proteins, including translation and transcription processes.

Transcription

• The turning of DNA information on how to make proteins into RNA in the form of mRNA, which is later exported from the nucleus

Translation

• The use of mRNA paired with tRNA by a ribosome to synthesize (make) proteins from amino acids

tRNA

• a type of RNA molecule

• coupled to an amino acid residue

• acts an adaptor, pairing with a sequence in mRNA to convert that codon sequence into specific amino acids

Ribosomes

• machines that catalyze protein synthesis

• composed of proteins and RNA, called ribosomal RNA (rRNA)

How does translation occur?

• Ribosomes incorporate specific amino acids into the growing protein using the genetic code

• There are 4 nucleotides and 20 amino acids

• By reading the sequence of RNA in groups of three consecutive nucleotide (codons), greater amount of information is conveyed in RNA (43= 64 combinations)

• Each codon codes for an amino acid, but different codons can code for the same amino acid

PET

• most common type of polyester

• an example of a polymer

• its monomer is ethylene terephthalate

• cooking it requires":

  • mixing

  • two precursors

  • Input of energy, heat (150-290oC)

  • removal of byproducts (such as methanol)

Special Amino Acids

• Ex:

  • Cysteine

    • contains a reactive sulfhydryl group (SH). Serves in catalysis of enzymes, reacts with other cysteines to form disulfide bonds to join two polypeptides

  • Glycine

    • smallest amino acid

  • Proline

    • has a side chain that forms a covalent bond to the N in the amino group attached to the alpha carbon kinks in the chain

Hydrophilic amino acids

• amino acids readily soluble in

water, “love water”

• either charged positively (basic), negatively (acidic), or polar (no charge)

Hydrophobic Amino Acids

• Amino acids poorly soluble in

water (hate water, fear water)

• either have hydrocarbons (linear or

branched) or aromatic rings

Nucleotides

• monomers of nucleic acids (DNA and

RNA)

• composed of:

  • pentose (five-carbon sugar), a phosphate group, and a ring-shaped molecule containing nitrogen called nitrogenous base (or just base)

• Negative charge in phosphate makes them acidic

What is the difference between DNA and RNA

• The difference between the two is

the loss of an OH group at position 2’

in deoxyribose

• The pentose in RNA (ribonucleic acid)

is ribose

• The pentose in DNA

(deoxyribonucleic acid) is 2’-

deoxyribose

DNA & RNA bases

two types:

• Purines, which contain a pair of fused rings

• Pyrimidines, which contain a single ring

• Thymine is only present in DNA

• Uracil is only present in RNA (replaces Thymine)

• Bases are connected to the ribose through the nitrogen atom at position 9 (in purines) or 1 (in pyrimidines)

• purine Adenine (A) pairs with the pyrimidine Thymine(T)

  • 2 hydrogen bonds (weak non-covalent bonds)

• purine Guanine (G) pairs with the pyrimidine Cytosine (C)

  • 3 hydrogen bonds

Nucleotide Ribophosphate backbone (DNA AND RNA)

• joined through two phosphodiester covalent bonds where a phosphate group links two (deoxy)ribose at postions 5’ and 3’

• Phosphate and (deoxy if DNA)ribose groups generate the backbone of nucleic acids

• DNA and RNA have an orientation, with a 5’ end (containing a phosphate) and a 3’ end (containing a hydroxyl group)

• Each monomer can contain a different base, hence, generating a unique sequence

Why is RNA more prone to breaking than DNA?

• Hydroxyl group at position 2’ in the ribose of RNA can spontaneously react with the phosphate group, break the RNA molecule

• DNA lacks an OH at the 2’ of ribose, making it much more stable and the choice for the genetic material in cells

DNA double Helix

• The ribophosphate backbone in the outside and

the bases inside

• Bases are regularly spaced (0.34nm appart)

• completes a turn every 3.4-3.6 nm (i.e.

10-10.5 bases per turn)

• On the outside, the spaces between the strands form two helical grooves of different widths:

  • the major and minor groove

• Atoms on the edges of each base are exposed

Major Groove

• on DNA double helix

• wide

• contains unique chemical information

• used by most protein-DNA interactions

Minor Groove

• on DNA double helix

• shallow

• cannot distinguish all bases

What evidence do we have that all life shares a common ancestor?

• Shared use of amino acids and nucleotides across life

• the use of only L-amino acid stereoisomers to generate proteins (not d-amino acids even tho theyre mirrorimages of each other)

• D-amino acids exist in life, but they serve other functions, such as during the cell wall synthesis in bacteria

Mirror Life/ Mirror amino acids

• ex: proteins composed of D-amino acids

• can have similar functions as their natural versions, but resist degradation by natural enzymes

• Potentially useful in clinic, but at the same time, controversial

What is the hierarchy of protein structure?

1. Primary structure

2. Secondary Structure

3. Tertiary Structure

4. Quaternary structure

Primary Structure (sequence)

• the first in the hierarchy of protein structure

• covalent bonding between amino acids

• Determines the size of a protein

  • expressed in the number of amino acid (aa) residues and their mass in daltons (Da) (1 Dalton=1 atomic mass unit)

  • Smallest proteins are ~40 aa in length

  • The ”average” protein depends on the organism. In yeast, the average is 466 aa residues. In E. coli is 329 aa. In humans is 476 aa

  • Largest protein known has 34,000 aa residues!

• Can form:

  • Oligopeptides = small chains < 30 aa

  • Polypeptide = larger chains, >30 aa and often hundreds of aa in length

Secondary Structure

• local conformations of the peptide chain backbone, generating stable arrangements of the aa residues

  • based on H-bonding between peptide bond carbonyl O atoms on one aa, and amide H atoms on another aa

• There are three major peptide chain backbone conformations:

  • α-helix

  • β-sheet

  • β-turn

• ~60% of the length of the average polypeptide chain consists of segments of α-helix or β sheet

• Several secondary structure elements per polypeptide

α-helix

• secondary structure option in proteins

• every NH of one amino acid at position n in the sequence forms an H- bond with the CO of a dif aa at n+4 position

• Tilted axis means there are 3.6 aa residues per turn

• Backbone has a straight rod structure with side chains pointing outward

• Side chains determine

  • the probability of it being formed

  • its interactions with other parts of the protein and other molecules

  • its surface properties (ex: polarity)

• Proline is not usually found in it as it creates a kink

β-strand sheets

• secondary structure option for proteins

• strands short (5-8 aa) nearly fully extended segments.

• H-bonds are formed between two adjacent β-strands perpendicularly to the backbone

• 2 or more strands generate nearly 2-dimensional sheets

• Side chains protrude above and below the sheet, determines:

  • interactions within the protein and with other molecules

  • likelihood of the sheet forming

• two different proteins can also interact to form sheets

• can be parallel or antiparallel

β-turn

• secondary structure option for proteins

• composed of 4 amino acids

  • forms a sharp U-shaped bend in the backbone

• usually contains glycine and proline

• The 1st and the 4th α carbon are held close by an H-Bond

• helps make protein structures more compact

Motif

• A combination of two or more secondary structures that form a distinct 3D structure

  • found in multiple proteins

• associated with a specific function

• ex: coiled-coil

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Coiled Coil

• 4 different α-helices coil about one another.

• aliphatic residues (hydrophobic but not aromatic) at positions 1 and 4 in a 7-aa repeat

  • these residues (aa) bury between the two helices.

• occurs in fibrous proteins and transcription factors

Tertiary structure

• overall conformation of the polypeptide (the spatial organization of the multiple secondary structure elements)

• stabilized by:

  • hydrophobic interactions

    • Water molecules surrounding hydrophobic molecules in an aqueous environment surround them, like a cage ( this has low entropy)

    • hydrophobic molecules group together (coalescence), reducing the amount of water molecules necessary to surround them

    • this net increase in entropy (less low entropy water molecules) causes separate hydrophobic and aqueous phases to form

  • van der Walls

    • favors coalescence of hydrophobic molecules (grouping)

  • hydrogen bonds

• these forces are weak, so the structure can fluctuate (change slightly now and then)

• divided in domains

Oil Drop Model

• a model for protein folding

• Hydrophobic and hydrophilic side chains are distributed throughout the linear sequence

• when the protein folds, the hydrophilic side chains tend to be exposed to water and hydrophobic residues tend to cluster together in the inner core, like drops of oil in water, hiding from the aqueous surroundings

• Within this simple general model an infinite number of particular structures are possible

  • but, in reality, each protein adopts only one or a small number of similar, structures (conformations).

Domains

• found in tertiary structures of proteins

• distinct regions

• They can represent a:

  • function

    • can be separated from the rest of the protein and remain functional

  • structure

  • or refer to the spatial relationship with the rest of the protein

    • It is common to refer to the regions near the

      ends of proteins as C-terminal or N-terminal domain

• similar versions can be found in different proteins

• multiple copies can be found in one protein

Green Fluorescent Protein (GFP)

• consist of a barrel containing 11 beta sheets and central helix that contains the chromophore

  • Interior of the barrel is highly crowded by side-chains of amino acid residues, limiting movement inside

• Alpha-helices at the ends of the protein stabilize it

• Chromophore (what gives the color) is formed by a post-translational modification that result in the cyclization of peptide backbone

Quaternary Structure

• Multimeric proteins several different tertiary structures held together

• can contain any number of identical or different polypeptides

• ex: Influenza virus hemagglutinin.

  • A trimer of identical subunits each composed of an HA1 and an HA2 domains

  • the three identical two-protein subunits are held together by non-covalent bonds

Supramolecular complexes

• Large “molecular machines” made up of multiple distinct proteins

  • each protein may itself contain multiple subunits.

• Ex: transcription initiation complex

How is X-ray crystallography used to determine protein structure?

• passes X-rays through a protein crystal containing millions of copies of the protein of interest in a precise arrangement

• X-rays are diffracted (change their trajectory) as they interact with electrons in the protein atoms

• this generates a pattern on the detector that represents the distribution of electrons (and atoms) in the sample

• Complex calculations are done to generate a model of the protein structure that matches the electron density.

How is Cryo-electron microscopy (Cryo-EM) used to determine protein structure?

• a solution containing the protein of interest is fast frozen on a grid.

• a small dose of electrons is used to prevent damage and detected using sensitive cameras.

• analysis sorts particles base on orientation to make average images and further detailed structures after further refinement.

How is AI used to determine protein structure?

• revolutionizing molecular biology

• sequence can be converted to a structure in seconds

• Capable of other predictions, like protein-protein interactions

• not 100% reliable, predicted structures provide a good model that can be tested by other methods

• how it works is unknown

Sequence Similarity

• degree of match between two sequences

• generally measured as a percentage

Homologous sequences

• two or more sequences derived from a common ancestral sequence

• either applicable or not, no middle ground

How to Identify Homologous Sequences

• Search for maximal similarity between sequence, aligning them such that as many aa’s as possible line up

• difficulty increases with length of sequence

• Introduce Gaps in the shorter sequence as necessary if there is a size difference between the compared sequences

• creating a dot plot representation can help

  • helpful to visualize changes like inversions and duplications

• Protein sequences contain more information on evolutionary conservation than DNA sequences

  • Conservation of chemical traits in different amino acids suggest evolutionary pressures to maintain activity

How does AlphaFold identify homologous sequences? What are its pros and cons?

• Protein query is compared with a database of protein sequences to look for local sequency similarity

  • This is then correlated to known protein structures

• predictions have low confidence when no sequence similarity is found

  • cause this is based on evolutionary conservation, and not on calculations of the chemical properties of proteins

• Protein structure prediction gets us closer to designing proteins with new functions

What is the order in which a protein begins to fold?

• 1st folds regions with secondary structure

• Secondary structure elements group into motifs …then into domains

• Finally, tertiary structure is acquired

• aka: same hierarchy as protein structure

How can amino acid residue rotate?

• the axis of the backbone can rotate at the bonds connecting the alpha carbon to the carbonyl and amide groups

• Rotation is limited due to steric constrains imposed by the backbone and the side chains

Describe the role Proline plays in folding

• Most amino acid residues result in a linear backbone

  • because they orient in a trans configuration relative to the peptide bond

• 5-7 percent of the peptide bonds with proline acquire a cis-configuration (creates a kink, no linear backbone

• Isomerization between trans and cis configurations can occur spontaneously, but it is slow.

• Peptidyl-proline isomerases (PPIases) are enzymes that speed up the process during folding

• Isomerization (cis to trans or vice versa swap) of a single proline can dramatically change protein structure, and affect its activity

Chaperones

• proteins that help guide protein folding along productive pathways

  • does so by permitting partially misfolded proteins to return to the proper folding pathway

• recognizes exposed hydrophobic residues.

• upregulated in conditions where misfolding is increased (ex. heat-shock)

• can:

  • fold newly made proteins

  • refold misfolded or unfolded proteins

  • disassemble potentially toxic protein aggregates that form due to protein misfolding

• works through ATP-dependent cycles of binding to, and release from, misfolded “client” molecules, at exposed hydrophobic patches

• By blocking the exposed hydrophobic patches, it isolates the folding or refolding protein while productive folding events occur.

• Two types:

  • chaperons (?!?!?!?, named this way because its the most common type so all of them are called chaperones 😕 , just use the e to differentiate for yourself they prolly wont)

  • chaperonins

Chaperonins

• large complexes that isolate unfolded proteins

• ex: GroEL

  • is composed by two stacked rings, each composed of seven subunits

  • Each ring interacts with a seven- subunit co-chaperone (GroES) that acts like a lid,

    • GroES also contains seven subunits

  • In the center of GroEL there are chambers where all or part of a protein enters.

Chaperons

• ex: Hsp70 ( Heat-shock protein)

  • Binding and hydrolysis of ATP are needed for Hsp70 as they result in changes in the conformation

• Binds to short segments of an unfolded proteins such as those newly synthesized as they emerge from the ribosome

If a protein is so misfolded that it cannot be fixed, then. .

• it is targeted for degradation

How does the Ubiquitin/proteasome system for protein degradation work?

• Poly-ubiquitin “tags” damaged or misfolded proteins for degradation

• Ubiquitin-tagged proteins are fed into a multisubunit chamber

  • subunits of chamber form inward-facing proteases.

• How it works:

  • 1. Ubiquitin ligases recognize exposed hydrophobic residues, and add multiple ubiquitins to proteins, forming chains of 4 or more ubiquitins.

  • 2. Polyubiquitinylated (chain of ubiquitin) proteins are recognized by Ub receptors in the proteasome

  • 3. Deubiquitinases (Dubs) hydrolyze bonds between ubiquitins to recycle them

  • 4. ATPase driven auxiliary proteins unfold targeted proteins and transports to the core for degradation

  • 5. Once inside the inner chamber polypeptides are digested into short fragments of 2-24 aa in length

  • 6.Peptide bonds of hydrophobic, acidic and basic residues are cleaved at the active site

  • 7.The resulting peptides are further degraded into single amino acids in the cytoplasm

Ubiquitin

• a small protein (76 aa residues, 8.6 KDa)

• becomes covalently linked to the lysine residue of target proteins

  • This requires multiple steps and a specialized machinery

• How it works:

  • 1. the carboxyl terminus is activated

  • 2. the protein is transferred to a ubiquitin conjugating subunit (E3 ubiquitin ligase)

  • (There are 600 E3 coded in our genome. Each with specific substrate binding.)

• This allows for it to mark proteins for degradation (after forming its own chain, aka its job in the ubiquitin/proteasome system):

Protein Aggregation

• 
  

• Formed when misfolded proteins or incompletely degraded proteins interact with each other, hiding their hydrophobic residues

• Occurs through high protein concentration and changes in environmental conditions

• can be amorphous (messy) or well- organized, such as in the amyloid state.

Amyloid Fibrils

• are formed by the generation of short segments (6-12residues in length) that form long arrays/ filaments, of β-sheets.

• Each β-strand is oriented nearly perpendicularly to the axis of the filament

• Two stacks pf beta strands twist about one another, forming protofilaments ( many of them form fibrils)

• can be found in tissues

• Neurodegenerative diseases such as Alzheimer’s disease and Parkison’s disease in humans, and the transmissiblespongiform encephalopathy (“mad cow” disease) in cows and sheep, all contain it

• its formation formation is associated to age, but more prevalent in mutant proteins

Ligand

• The molecule to which a protein binds

• Diverse functions performed by proteins are based on their to engage in a common activity: binding

• Proteins bind to one another, to other macromolecules, to small molecules, and to ions.

• Two important properties in protein binding:

  • Specificity: the ability of a protein to bind only one particular ligand, even in the presence of a vast excess of irrelevant molecules

  • Affinity: the tightness, or strength of binding, expressed as dissociation constant (Kd). The stronger the interaction, the lower the Kd

• Protein binding results from an interaction between complementary molecular surfaces

• Protein binding specificity arises from interactions which are individually weak, but, if numerous, collectively strong

Antibodies

• contains two (identical) heavy chains and two (identical) light chains.

• Individual polypeptides (chains) in it are kept together through disulfide bonds

• Molecules it recognizes are called antigens

• The antigen-binding surface or CDR (complementarity determining region) involves multiple protein loops from both the heavy and the light chains

• These loops are highly variable in amino acid sequence among antibody encoding genes so the total repertoire of possible CDRs in the antibody population is huge (lots of variety)

Enzymes

• (proteins or RNAs)

• catalyzes the making or breaking substrate covalent bonds

• Ligands of enzymes include the substrates of the reactions they catalyze

• accelerates the rate of a reaction by reducing the free energy of the transition state and thus ΔG‡.

• Substrate specificity arises from the substrate binding site

• The substrate, and the substrate binding site interact at complementary molecular surfaces.

• Some amino acids in the enzyme polypeptide contribute to the substrate binding site, and others contribute to the catalytic (active) site

• Vmax – the maximal rate of catalysis given saturating amounts of substrate

  • Depends on the amount of enzyme and how fast it can work:

    • the turnover number

    • and the enzymatic cycles per second at top speed

• Km – the substrate concentration that supports a rate of catalysis equal to one-half of the Vmax

  • Provides a measure of the enzyme: substrate binding affinity

• Enzymes free in solution: reaction intermediates diffuse from one enzyme to the next

  • can be slow. .

• Coupling by a scaffold protein overcomes slow substrate diffusion in a metabolic pathway

• Multisubunit enzyme complex formed by a scaffold protein minimizes or eliminates the substrate diffusion time

• Some enzymes are fused at the genetic level, becoming domains in a single polypeptide chain

Serine protease trypsin

• Proteases hydrolyze peptide bonds in polypeptides.

• The serine proteases are a family of proteases whose catalytic mechanism involves a serine residue in the catalytic site.

• Trypsin hydrolyzes peptide bonds adjacent to arginine and lysine (large, basic side chains).

• Proper substrate binding only occurs when the substrate amino acid side chain “fits” into a negatively charged pocket within the substrate binding site

• Differences in the substrate recognition pocket in the related enzymes define their differing specificities.

  • ex: The pocket in elastase is obstructed by bulky valine side chains, so elastase cleaves adjacent to amino acids with small side chains, e.g. alanine and glycine

• How Trypsin’s (serine protease trypsin) Catalytic site works:

  • Protein folding brings “distant” amino acids into proximity.

  • Trypsin uses a 2-step catalytic mechanism involving 3 amino acid side chains: Asp-102, His-57, and Ser-195

    • Step 1 (a-c) cleavage of peptide bond with formation of a covalent substrate-enzyme complex (Ser195 acyl enzyme).

    • Step 2 (d-f) hydrolysis of acyl enzyme complex

    • Both subreactions depend on His-57’s ability to bind, and to release a proton.

    • These reactions happen best at a pH near the pK of His, i.e. ~ pH 7.

      
      

      
      

pH requirements for Acid-Base Catalysis

• Catalysis requires a particular ionization state (protonated or nonprotonated) of one or more amino acid side chains in the catalytic site.

• Pancreatic serine proteases (chymotrypsin) have pH 8 as activity optimum

• pH <9 for required for proper protein conformation

• Lysosomal hydrolases ∼4.5 pH optimum: matches the low internal pH in the lysosomes in which they function

Allosteric Regulation

• A major manifestation of allosteric effects

• conformational switches in regulatory proteins in response to ligand binding or post-translational modification

• Allosteric Effects result from the binding of a ligand at one site on a protein, which leads to conformational changes that affect the binding of another ligand molecule at a different site of the same protein

• ex: binding of ATP in chaperones,

  • Ca++ binding to calmodulin changes its conformation, allowing it to bind to target peptides on other proteins, thus regulating their structure and activity

  • G-proteins exist in “on” (GTP bound) and “off” (GDP bound) conformations that interact differently with other proteins

    • Switching from “on” to “off” is facilitated by GAPs and switching from “off” to “on” is facilitated by GEFs

      
      

      
      

Note: Subcellular localization also helps regulate protein function

Post-translational modification of proteins

• changes in the chemistry of proteins that occur after synthesis (ex. ubiquitinylation)

• Modification of protein conformation and activity can occur through phosphorylation and dephosphorylation

• Protein kinases catalyze the phosphorylation of amino acid side chains

• Protein phosphatases catalyze dephosphorylation of amino acid side chains

• Human genome encodes >500 different protein kinases!

• Kinases recognize a sequence in proteins and introduce a phosphate group at serine, threonine or tyrosine.

  • The reaction transfers the gamma phosphate in ATP to the protein modified

Purification of Proteins

• Proteins in our cells vary widely in their properties (size, charge, hydrophobicity).

• These properties are exploited for their purification.

• Proteins are normally extracted from cells, by breaking them and removing big cellular debris (like membranes, RNA and DNA).

Chromatography

• Separation of components based on their differential interactions with an immobile (=solid) material.

• Mobile phase (liquid) moves continuously past the solid phase (paper in the experiment we did in lab).

• Protein molecules are moved along in the mobile phase but at rates that depend on how much they interact with the solid phase.

• generally done in columns, with a solid phase made up of ~millimeter-sized beads

• 3 types:

  • gel filtration

  • ion exchange

  • antibody affinity

Gel Filtration Chromatography

• Separation based on size and shape.

• The solid phase gel beads have pores (tiny, made for molecules)

• The mobile phase freely enters and exits the beads (flows through)

• Small proteins can enter and exit the beads through the pores (a waste of time going in and out)

• Large proteins cannot enter.

  • They don’t “waste time” diffusing within the beads, so they migrate faster and are eluted earlier.

• Shape also influences migration.

  • The closer to spherical the shape, the slower the migration

Ion-exchange chromatography

• Separation based on electric charge

• Protein molecules with a net electric charge will bind to immobile phase with the opposite charge (and thus will move slower, so whatever molecule has the same charge as the immobile phase will move faster down the column, as they will be repelled)

• Electrostatically-bound proteins can be released by flowing a salt solution through the column (to collect them after the faster ones have already been collected)

• The displacement of the protein by Na+ or Cl- is “ion exchange” (from the salt being flown down)

  
  

  
  

Antibody affinity chromatography

• Antibodies specific for any particular protein can be covalently coupled to the solid phase

• All the other proteins can be washed away and then the target protein can be released from the antibody by lowering the pH

• bypasses all other properties of the protein purified, as long as it can bind to the antibody (or other molecule) in the solid phase it will work

electrophoresis

• In a free solution at a given electric field, the direction of migration is determined by the net charge, and the speed of migration is determined by the net charge/mass ratio.

• Protein mixture is loaded at the wells of gel (made of cross-link/bonds of the polymer polyacrylamide)

• Proteins migrate through small pores in the polymer network.

• Smaller proteins migrate faster

• In native proteins, migration depends on charge, size and shape

• Since the start point has a negative charge and end point has a positive charge, negatively charged proteins move faster (repelled by start attracted by end) than positively charged proteins

SDS-PAGE

• Anionic detergent sodium docecyl sulfate (SDS).

• SDS denatures proteins by the interaction of its hydrophobic tail with hydrophobic amino acid side chains, disrupting the oil drop structure (hydrophobic inner, hydrophilic outer) of proteins

• The hydrophobic tail of SDS binds not only to hydrophobic residues, but also to other SDS molecules,

  • so it tends to coat the polypeptide chain in auniform layer of SDS molecules

• Because these are all negatively charged, the various parts of the SDS polypeptide chain repel each other and this further disrupts and unfolds the protein

• SDS-PAGE was developed to study of the proteins of a phage. Progress in science cannot be predicted!

• Denatured proteins (proteins that lost their structure) covered by SDS are separated by size through electrophoresis (all are negative due to SDS coating)

  • They migrate towards the positive pole

• All proteins are then coloured by using dyes that bind to them non-specifically

• A pattern of bands, each band representing a different protein, can be observed

• SDS-PAGE is normally used AFTER protein purification to assess degree of purity (it is not a purification technique)

• Alternative specific proteins can be detected by the use of antibodies

• They are then Immunoblotted

  • All proteins in an SDS-PAGE gel are transferred to a membrane, where proteins will be exposed in its surface.

  • Proteins can now be accessed by antibodies.

  • Normally, a primary antibody detects the protein of interest.

  • A secondary antibody, used for detection through fluorescence or enzymatic activity, is used to detect primary antibody.

  • Hence, only few ‘detection’ antibodies are needed

Immunoblot (western blot

• How it works (using SDS):

  • All proteins in an SDS-PAGE gel are transferred to a membrane, where proteins will be exposed in its surface.

  • Proteins can now be accessed by antibodies.

  • A primary antibody detects the protein of interest.

  • A secondary antibody, used for detection through fluorescence or enzymatic activity, detects the primary antibody.

  • Hence, only few ‘detection’ antibodies are needed

• Density of bands represent protein abundance

• Another Example:

  • Experiment tests whether the presence of the G (glucocorticoid) R ligand influences the abundance of its receptor

  • Detection of actin, serves as reference for quantification (loading control

Co-immunoprecipitation

• Use of an antibody that recognizes a specific protein to co-purify other proteins that interact with it

• This technique can be used to identify proteins in a protein complex

• The immunoprecipitate will contain the protein carrying the epitope (part of an antigen recognized by the antibody) and any partner proteins stably associated with that protein. (i.e. co-immunoprecipitation) (co-IP).

• ex: (diagram) is testing the interaction between GR and PPARa, and the effect of GR-ligand in the interaction

  
  

Immunofluorescence

• identifies cellular localization of proteins

• Localization of proteins in cells and tissues studied by the use of antibodies

• How it works:

  • Cells are killed using solvents that preserved their structure and immobilize on a microscope slide

  • Antibodies are then used to detect specific proteins,

  • Fluorescently labelled secondary antibodies are then used to mark the original antibodies (done at the fluorescence microscope)

Fluorescence

• the property of certain substances of absorbing light of short wavelength and emitting light of longer wavelength

• How it works:

  • An electron in the molecules that produce fluorescence (called fluorophores) moves to a higher energy orbital as result of the absorption of a photon (Excitation)

  • Excited electrons have a tendency to go back to their initial ground state

  • In doing so they release the excess energy in the form of light (Emmision)

  • Fluorophores (chemical compounds that have fluorescence) undergo thousands of excitation-emission cycles before they are chemically modified (oxidized) and stop producing fluorescence (Bleachin)

• Fluorescence Microscope

  • 4 important elements

    • 1. Light source capable of producing intense light over a broad spectrum

    • 2. Filter Cube

      • contains an emission and excitation filter

    • 3. Objective lens

    • 4. Sensitive camera (similar to those used in Astronomy)

  • also has a Dichroic mirror: Reflects one wavelength and transmits another (look at diagram lowk)

• first described in jellyfish and coral, now we know it is widely distributed in animal species

• How its used to label a protein of interest:

  • Gene fusions are generally done by cloning before being introduced to the organism with either:

    • a vector or by homologous recombination (to insert them in the genome).

  • Expression results in fluorescent protein fusions

  • Choice of C- or N-terminal fusion depends on the structure and activity of the protein of interest

  • Multiple colors, from blue to far-red are possible

  • Multiple fluorescent proteins can be combined in a single sample.

  • In contrast to immunofluorescence, where cells are dead, use of GFP allows real-time tracking of cellular events

DNA Replication (Simple basic og slide on it)

• Parental strand can be used as a template for the formation of a new daughter strand (complementary to the parental strand)

• Phosphate group in the incoming deoxy nucleotide triphosphate (dNTP) reacts with the 3’ hydroxyl group (OH) in the growing DNA chain

  • simplified: phosphate in the new nucleotide added to the growing strand binds to the 3’ hydroxyl group (OH) of the previous nucleotide to become a part of the growing DNA (phosphate becomes part of growing backbone)

• The opening of the DNA duplex for replication occurs progressively in cells, forming a DNA structure called the replication fork

Replisome

• the Molecular Machinery that carries out DNA Replication

• Different parts:

  • DNA Helicase/ CGM Helicase

    • separates the strands in order for duplex DNA to be replicated

    • Hexamer of MCM

    • Contains accessory subunits Cdc45 and the GINS complex

    • Binds to the leading strand

  • Replication Protein A (RPA)

    • binds to single-stranded DNA

    • keeps single-stranded DNA template in optimal conformation for DNA polymerase

    • Prevents formation of secondary structures on single-stranded DNA

  • Primase/Pol alpha Complex

    • primase forms the RNA component of the primer

    • and DNA polymerase a (alpha) (Pol a) extends the primer with DNA

  • Pol δ replaces the Pol α/primase complex for the lagging strand and completes the synthesis of an Okazaki fragment

  • NOTE IMPORTANT: Pol α/primase is the primase you learned in school, it DOES NOT synthesize strands, its RNA POL, Pol ε synthesizes the leading strand and Pol δ the lagging strand

  • PCNA

    • homotrimeric protein.

    • Prevents the Pol ε (or δ) complex from disassociating from the template

  • Rfc/PCNA complex

    • RFC is the PCNA loader

    • it opens the PCNA ring and loads it at a primer on DNA

  • Ribonuclease H and FEN-1 displace the RNA component at the 5’ ends

    of the Okazaki fragments.

    ➢ Pol δ replaces the RNA with DNA.

    ➢ DNA fragments are then ligated together by DNA ligase

• Tightly regulated

  • Origin Recognition Complex (ORC) is a six subunit protein that

    marks replication origins and loads helicase aided by other initiation factors

  • MCM helicase is loaded in G1 phase of the cell in an inactive form

  • MCM is activated in S-phase through phosphorylation and interactions with other proteins

  • CDK = cyclin dependent kinase

    • Low CDK levels in G1 → helicases is loaded but not yet activated

    • High CDK levels in S-phase → no loading of helicase (already loaded), helicase is activated

Supercoiling

• Opening of the double helix causes an increase in the twist of DNA ahead of the replication fork

• Both, under and over twisting the DNA is called supercoiling (negative or positive, respectively).

• introduces torsional stress and deforms DNA.

• Accumulation can inhibit DNA replication and transcription

Topoisomerase

• removes supercoiling ahead of the replication fork

• works by transiently cutting DNA

• For example, Topoisomerase II, cuts a double stranded DNA to allow a second piece of DNA pass through the opening

DNA Polymerization

• catalyzed by DNA polymerase

• requires deoxynucleoside 5’-triphosphates (dNTPs, where N = A, T, C or G)

• requires a primer (can be DNA or RNA)

• proceeds from a 5‘ to 3’ direction

• Free dNTPs are added through the formation of a new phosphodiester bond involving the 3’ hydroxyl of the terminal sugar

• Primase (a specialized RNA polymerase) forms a short RNA molecule complementary to a single-stranded region of the unwound duplex DNA

• DNA polymerase extends the primer to eventually form a new daughter duplex DNA.

• Since the primer is RNA then the addition of dNTPs by DNA polymerase will result in a molecule that is RNA at the 5’ end and DNA at the 3’ end

• DNA replication proceeds in a 5’ to 3’ direction and the two strands of duplex DNA are antiparallel.

  • PROBLEM: Although one new strand (called the leading-strand) can be replicated 5’ to 3’ by DNA polymerase following the movement of the replication fork, the other new strand (called the lagging-strand) must be formed in the opposite direction of the movement of the replication fork

  • SOLUTION: Okazaki fragments are formed

    • Okazaki fragments are short discontinuous fragments consisting of RNA and DNA

    • They are formed on the lagging strand as many RNA primers are needed to allow for it to be synthesized with DNA Pol δ, and so it creates a mix of DNA and RNA when Pol δ places DNA between them (Pol δ replaces the Pol α/primase complex for the lagging strand and completes the synthesis of an Okazaki fragment)

    • The RNA component of the Okazaki fragments are then replaced by DNA and the two adjoining DNA molecules are ligated together by DNA ligase

      • (Ribonuclease H and FEN-1 displace the RNA component at the 5’ ends

        of the Okazaki fragments.

        ➢ Pol δ replaces the RNA with DNA.

        ➢ DNA fragments are then ligated together by DNA ligase)

        
        

DNA Pol Epsilon

• Carries out leading strand DNA synthesis

Origins of Replication

• Specific region(s) at which assembly of the replisome and initial unwinding of DNA occurs in duplex DNA.

• tends to be AT-rich (Adenine and Thymine)

• Replication proceeds bidirectionally from the origin

  • unwinding by the CMG helicase is driven by ATP hydrolysis

  • RPA binds to single-stranded regions

    
    

• Some of the replication origins in our genome are activated first (called early origins) resulting in the initial replication of some regions of the genome

• As S phase progresses, other origins activate, following a ‘program’

• The replication program is established early during development and is cell type- dependent

• The replication program is related to transcription and is important for genome integrity

• Our genome is composed of 3.3 billion base pairs

  • At a rate of 2,000 bp / minute, a single replisome would take ~ 3 years to complete DNA replication

  • Hence thousands of replisomes originating from tens of thousands of replication origins are used at each cell cycle

  • This reduces the replication time in somatic cells to few hour

Mutations

• If the changes these cause are permanent, they are mutations :

  • 2,000 – 10,000 hydrolytic depurination every day / cell

  • ~1 cytosine deamination every 5 days/cell

  • ~1 event of Guanidine oxidation (8-oxo-guanine) every 5 days / cell

  • ~600 events of methylation of adenine (3-meA) per day/ cell

  • Some of these modifications alter the original base pairing and may also block the activity of DNA polymerases use during DNA replication

  • DNA polymerases alone introduce 1 error every 10,000 incorporated nucleotides.

  • Proofreading exonuclease activityincreases fidelity by ~100 fold (1 in every million)

• permanent, transmissible changes to the genetic material of a cell (or organism)

• can occur spontaneously, by transposable elements, and by errors during replication

• Mutagens are chemical compounds that increase the frequency of mutations

  • ex:ultraviolet (UV) radiation or ionizing radiation (e.g. x-rays and atomic particles)

• Carcinogen is an agent that causes cancer. Many carcinogens are mutagens

• The number of mutations that fathers give to their offspring increases with age due to continuous production of germ cells (at about 2 additional per year)

• A curious case

  • T. dohrnii (a.k.a. immortal jellyfish) can revert to a sexually immature polyp stage in response to stress or ageing

  • Its genome contains multiple copies of MSH2 and of DNA Pol alpha and delta, which was suggested to lower its mutation rate in somatic cells

Proofreading & Repairing DNA

• In eukaryotes, DNA polymerases ε and δ but not α have a 3’ to 5’ exonuclease or “proofreading” activity

• Mismatch repair further increases fidelity by about 1,000 fold, reaching error rates of about 1 error in every 1,000,000,000 nt incorporated.

  • Adding it all, there is a probability of incorporating a single mistake of 10^9-10^10 per nucleotide in human cells

• Multiple mechanisms act to repair specific types of DNA damage

  • Mismatches and chemical modifications in DNA are repaired by a strategy that requires the detection and removal of the affected region

    • The second strand is then used as a template to fill the ssDNA gap by a DNA polymerase

  • Mismatch Repair (MMR) (In Humans)

    • Error incorporated into newly synthesized strand (daughter strand), error is wrong base pairing (ex: A and C)

    • MSH2 and MSH6 bind to errored daughter strand

    • This triggers binding and activity of MLH1 endonuclease (dimerized w/ PMS2), which make cuts near the mismatch

    • DNA helicase unwinds and DNA exonuclease digests the cut segment of daughter strand

    • The gap left behind is repaired by Pol δ and DNA ligase

    • often acts in proximity to the relisome, correcting mismatches shortly after they have been generated

    • Some of the proteins involved in MMR interact with PCNA (MSH2 and MLH1)

  • Base Excision Repair

    • How it works:

      • DNA glycosylase hydrolyzes the bond between the mispaired base and the sugar-phosphate backbone

      • In the case of uracil removal, the DNA glycoysylase is called UNG

      • Apurinic/apyrimidinic endonuclease 1 (APE1) cuts the DNA backbone

      • AP lyase associated with DNA polymerase β removes the deoxyribose phosphate (backbone that was cut)

      • DNA polymerase β fills the gap and DNA ligase seals together the sugar-phosphate backbone

    • many types of base modifications possible, 11 different DNA glycosylases specifically recognize some of these types of base modifications to start repair

      • ex: deamination, oxidation, alkylation

  • Repair of bulky lesions (Nucleotide Excision Repair)

    • ex: Formation of thymine-thymine dimers in DNA (caused by UV lighting on outer thymines that just happened to be next to each other)

    • BIG problems in DNA, bulky issues

    • Can be caused by:

      • Food contaminated with fungi (for ex Aspergillus)

      • Smoke from organic material, such as cigarette smoke and charbroiled food

    • How it works:

      • A complex formed by proteins 23B and XP-C do the initial recognition of the problematic DNA lesion

      • The complex of 23B and XP-C recruit the transcription factor TFIIH, which catalyzes the unwinding of the DNA region around the lesion

      • This in turn recruits RPA and XP-G, which also has unwinding activity (also unwinds DNA)

      • Finally, XP-F is recruited.

      • Both XP-F and XP-G have endonuclease activities

      • They generate cuts 24-32 bases apart, releasing the produced fragment carrying the lesion (problem) to be degraded

      • The ssDNA gap generated is then filled by a DNA polymerase and joined to generate a continuous strand by DNA ligase

    • Translesion Synthesis

      • Note:

        • DNA repair mechanisms require that DNA is in a double-stranded form

          • During DNA replication, the replisome may encounter lesions on DNA that were not previously repaired

          • The DNA polymerase in the replisome will stop when it encounters this, preventing completion of DNA replication

      • Cells use a special type of DNA polymerases called translesion (TLS) polymerases

      • TLS polymerases are able to bypass the lesion (ignore the problem and keep synthesizing), using the damaged DNA as template

      • However, they will often insert the incorrect base at the position opposite to the lesion.

      • TLS do not have proofreading exonuclease activity. They are error prone.

      • In the end, the DNA lesion is left in the template DNA

Breaking in DNA strand

• Single strand breaks are more common than double strand breaks

  • and are efficiently repaired by the ligase

• DNA unwinding during DNA replication can convert a single strand break into a double strand break

• Repair: Error-prone repair by non-homologous end- joining (NHEJ) or repair by homologous recombination (HR)

  
  

  
  

Singe StrandHomologous Recombination

• YOU HAVE TO LOOK AT DIAGRAM, I TRY TO WRITE DOWN BUT ITS HARD

• How it works:

  • 1. After the break, the replication fork collapses, the separated bits stay with the newly made strands, the ones that were not open yet (not a part of the fork yet) stay together, creating a mix and match of strands

  • 2. 5’ exonuclease acts on the broken end (of the new strand without continuation since the replication fork broke), the other daughter strand (one with continuation) is ligated to repaired parental strand in the unbroken chromosome (to the portion that was outside of the fork)

  • 3. recombinase RecA or Rad51 mediated strand invasion takes place ( the new strand that got ligated together with the unopened dna strand lifts closer to the other new strand and away from the unaffected original strand it was partnered with, invading that space, the discontinued original strand/the other strand is now under it and closer to the original strand)

  • 4. Branch Migration ( The branches criss cross to create Holliday Structure, the broken/affected original strand moves down and crosses with the lifted strand)

  • 5. Cut strands at cross-over (the holliday structure is cut from the middle of the x)

  • 6. Ligate ends (put closeby broken bits together to form mixed strands)

  • 7. Rebuild Replication fork and continue DNA replication

Double Strand

• High fidelity mechanism to repair DNA breaks

• Requires an undamaged copy of the chromosome to be repaired

• The process requires:

  • processing of breaks to expose ssDNA sequence,

  • a search for identical sequences,

  • using said identical sequences as template of DNA synthesis ( to repair the break)

• How it works (PLEASE LOOK AT DIAGRAM THIS IS SO HARD TO JUST WRITE):

  • 1. Ends digested by 5’-exonucleases leaving behind 3’ single stranded ends (on each opposing side of break one of the 2 strands (different on each side) has part of it eaten)

  • 2. recombinase RecA or Rad51 mediated strand invasion of homologous chromosome (one of the uneaten longer ends moves toward the undamaged copy double strands and lifts one above itself (doesn’t have to be lift above it’d be based on pov but thats how the diagram shows it, basically swaps positions without breaking) to create an X, a Holliday structure technically but not mentioned yet)

  • 3. 3’ end of the invading strand is extended by DNA polymerase until the displaced single-strand base pairs with the other 3’ single strand generated initially (this one is pretty nice explanation, the one that lifted keeps lifting toward the 3’ end until the strand its lifting base pairs with the other broken strand )

  • 4. 3’ end is extended by DNA polymerase (of the other broken strand that was mentioned to have base paired)

  • 5. ends are ligated to form holliday structures (X’s are formed from the lifted and the lifter (2 X’s including the one from earlier)

  • 6.Holliday structures are resolved (the X is broken and the crossed strands mix together to form 2 mixed strands each)

• Two Holliday junctions have to be resolved during Homologous Recombination

• Alternative Resolutions are possible (need to show diagram so its on a different card)

• Each Holliday junction can be resolved at any of the two orientations

  described previously (on the other card)

• Their combination determines if they final result is a recombinant chromosome (crossover)

• During meiosis DNA recombination occurs between chromosomes from mom and dad to mingle their sequences.

• Sequences of father and mother derived chromosomes are mingled, producing translocations between homologous chromosomes

Alternative Resolution of a Holliday Structure

• PLS LOOK AT DIAGRAM

• 2 WAYS TO RESOLVE

  • 1. the original holliday strands break & reconnect (Original)

  • 2. outer strands break and reconnect to mixed holliday strands allowing for recombination (Recombinant)

Non-Homologous End Joining (NHEJ)

• Error-prone and can introduce mutations

• requires blunt ends for ligation

  • Hence, exonuclease and DNA polymerase activity is used so that all nucleotides are paired with the complementary strand.

  • This results in a loss of information, either by the removal or the addition of nucleotides.

• How it works:

  • A complex formed by DNA-dependent protein kinase (DNA-PK) and Ku80/Ku70 heterodimer binds to the ends of the break

  • Ends are digested by nucleases or filled in by DNA polymerases

  • Ends are ligated together

• A chromosomal translocation is an abnormal chromosome region that contains rearranged genetic material.

  • In NHEJ these comes from two non-homologous chromosomes.

  • Since NHEJ joining of DNA fragments is not driven by sequence similarity, these can result in mistakenly bringing together pieces of two very different chromosomes

In what part of the cycle does each type of DNA repair occur?

• During G1, where only one copy of the genome is present, DNA breaks are repaired by NHEJ

• During S and G2, and M phases, double strand breaks are repaired by Homologous Recombination

• During S phase, Mismatch Repair and Translesion Synthesis occur

DNA repair and Cancer

• Breast Cancer

  • Breast cancer type 1 susceptibility protein (BRCA1) binds to double strand ends and promotes their resection.

  • BRCA2 acts during recombination to promote the loading of Rad51 onto the resected DNA.

  • Mutations that inhibit the function of these proteins are associated with a predisposed development of cancer, particularly breast and ovarian.

    • This is likely due to an increased used of NHEJ for DNA break repair.

  • PARP inhibitor (PARPi)

    • acts by blocking the repair activity of single strand DNA discontinuities

    • During DNA replication, this would generate double stranded breaks

    • A high number of DNA breaks in cells without active DNA recombination (because of breast cancer, BRCA1&2 aren’t able to function to fix it) results in high NHEJ activity, generating too many issues in the genome and leading to cell death

    • There are other models proposed to explain the mode of action of PARPi

• Cells in only few tissues actively divide, like skin cells, cells in the gastrointestinal tract, and blood cells in the bone marrow.

• In most other parts of the body, cells do not divide often or at all, like in the brain and the muscles.

  • This contrasts with tumours, where cells constantly grow and divide.

    • This is why, treatments that damage DNA are effective against tumours

• Many of the drugs and treatments used for anti-cancer therapy target DNA.

• DNA damage can be repaired efficiently if DNA replication is not ongoing.