Bio 110 Exam #2

The Problem with Kornberg's Polymerase? (DNA Polymerase

-1969 - Mutants lacking DNA Pol I are viable!!! *geneticist took E. coli, identified gene for polymerase I, & removed gene; created bacteria w/o gene for polymerase that Kornberg found & it was viable *implications: viable & can reproduce w/o polymerase (polymerase I NOT responsible for copying chromosome like Kornberg thought!!) -DNA Polymerase I is not (alone) responsible for chromosomal replication -Kornberg's Polymerase is for Repair! (look for mistakes & repair them) -E. coli has 5 DNA polymerases (DNA polymerase III is the main polymerase responsible for chromosome replication)

Mucin

-3 other features of protein that form linkages btwn diff units (besides sugar interacting w water & more sugar, glycoprotein): 1. dimerization (dimer=2 units) domain: glue together copies of protein using dimerization domains, stick to another copy of same domain (bind non-covalently) 2. have lots of cysteines throughout structure: goal to make lots of connections btwn diff copies of protein; make disulfide bonds (covalent) 3. small, globular domains: protein largely has regions unfolded, but small regions fold into globular structure w hydrophobic core; if become denatured & expose hydrophobic residues, hydrophobic residues cause them to clump together **when sick, pH lower slightly & provides additional way for mucins to cluster together -mucin subunit->mucin dimer->mucin tetramer->mucin networks (w higher order complexation), results in formation of mucin aggregates -Mucin aggregates invariably contain two-distinct zones: the more intact adherent mucin layers, and loosely-held (expanded) mucin layers of high free-volume *in pic, all hairs coming off are carbs

Adenine in lots of important places (1 of our bases & the A in ATP)

-ATP is one of our 4 bases we make DNA out of; can use other bases to get energy just like ATP (like GTP) -cyclic AMP: regulating signal transduction & gene expression -ATP has adenine -FAD & NAD: electron carriers -coenzyme A -caffeine mimicking adenine & interferes w many of these molecules in diff places (that's why it has pleasant effects)

The Central Dogma (Francis Crick)

**tells the flow of information DNA (DNA replication) -transcription->RNA-translation->Protein -can have reverse transcription RNA->DNA -circular arrows (both DNA & RNA): can use DNA as template to make new DNA -some viruses replicate do same thing, replicate RNA genome -RNA -> proteins is a one-way street

Hemoglobin is allosteric

*allosteric=change shape, has 2 distinct configurations: T & R (diff functions) *if O2 binds to heme ring in 1 of 4 subunits, can change structure of that subunit; since subunit bound to other subunits, changing it will change the structure potentially of whole molecule -When Oxygen is bound, the shape of the entire peptide changes slightly - beginning with the movement of the iron - it becomes on the same plane as the ring holding it. -The change in one peptide changes the shape of the other peptides. -allosteric = changes shape (other + shape) -hemoglobin can take more than 1 shape

The binding of O2 to hemoglobin causes a slight shape change

*within protein, histidine attached to alpha helix! *if O2 binds, pulls up on histidine & bends alpha helix -> that's how the structure of that subunit changes *this breaks a few bonds & changes structure of other subunits *when O2 binds even 1 subunit, can change structure of whole molecule of hemoglobin -Changes in peptide shape changes binding btwn peptides (due to changes in ionic attractions). The resulting shape changes in other peptides create confirmation w higher affinity for O2. = Cooperative binding -without O2 <--(low pH, high CO2)-- with O2 -lower affinity for O2 (not sticky for O2) --(O2)--> higher affinity for O2 (really wants to bind) *pH of lungs higher & high O2 concentration *even in low affinity state, we increased O2 conc & probability of binding; as long as bind O2 to 1 of 4 subunits, rest of it turns into high affinity state & can quickly grab 3 more O2 *so this protein is adjustable in terms of its structure & function; can dial up affinity for O2 when need to in lungs where O2 conc high (pick up 4 molecules of O2) -> then travel to muscles: O2 conc low, more likely to release molecule of O2 -> then move back toward low affinity state & drop off rest of O2 **so it's an adjustable protein due to O2 concentration

Other weak forces that help proteins fold (start L2)

-Although a single one of these bonds is quite weak, many of them often form together to create a strong bonding arrangement. -denature: normal structure of protein disrupted (not just shape); function also destroyed *denature protein w temp (breaks hydrogen bonds), pH (prevent ionic interactions; change pH changes charge of diff regions of protein, charge imp for holding protein together; ex added protons bond w neg charged things to make neutral), etc -hydrogen bonds, ionic interactions, van der Waals

The Problem w Water When It Freezes

-Crystal growth of pure ice: a) seed ice, b) ice crystal grown along the a-axis, c) hexagonal ice crystal formed, d) ice crystal further grown from the edges of hexagonal, e) star like ice crystal formed, and f) ice sheet started to form. -How can proteins do this? -Ice crystal growth with antifreeze protein - in presence of 6 mg/ml type I Antifreeze protein (AFP) *antifreeze proteins prevent growth of small ice crystals into bigger ones (cells don't burst open)

DNA Structure

-DNA is a double helix -phosphate groups neg charged, on outside of DNA & makes it soluble (if packed in center would repel each other)

Unfolding/misfolding Proteins

-Denaturing agents destroy the protein tertiary structure (causes the protein to begin to unfold). -some Methods: (how do these work?) • Heat (break hydrogen bonds) • Extremes of pH (add/remove H+ changes charge of diff regions of protein; prevent ionic interactions) • Detergents (amphipathic): similar to fatty acid structure (long hydrophobic tail & hydrophilic region on end) *denatures bc hydrophobic regions of detergents interact w hydrophobic regions of protein & disrupt structure • Mechanical agitation • Urea: interfere w hydrogen bonds • Mercaptoethanol: breaks -S-S- bonds (disulfide bonds) **once denature protein & remove denaturing agent, protein can't always go back to native structure (can't uncook an egg) -What is exactly happening when a protein is denatured? -why do denatured proteins tend to be less soluble than the native protein? can it be renatured?: cluster of hydrophobic regions=stable

Other problems - how to deal with then ends of linear chromosomes?

-Disadvantage of linear chromosomes: ends -Problem: With each replication the ends get shorter. -Solution: Telomerase -replication w circular chromosomes easy bc don't have ends; ends of chromosomes difficult to replicate completely bc need to start replication w RNA primer then remove RNA primer -as get closer to end of chromosome, if on v end of chromosome, need to remove primer & there's no space off end of chromosome to put new primer -if didn't have a way to fix prob, would lose small part on end every time replicate linear chromosomes bc small piece remains single-stranded, unreplicated -want to fill in that space, only have enough room for primer; leaving end of chromosome single-stranded bad bc would get chopped off (single-stranded gets degraded, would start destroying genes)

How Base Pairs are Pairing in Center of Double Helix

-Distance for purine + pyrimidine is similar regardless of pair - how would a mismatch compare? -3 H bonds btwn G & C, 2 btwn A & T -A has 2 rings, T has 1; C has 1 ring, G has 2 *distance btwn these backbones always approx the same (have 2 ring base & 1 ring base) *if add wrong base, shape of double helix either too wide/too narrow depending on mistake (how we detect mistakes when making DNA); won't have nice uniform distance btwn backbones if add the wrong base

DNA can vary in its overall configuration

-Franklin found that DNA can take on more than one shape (depending degree of dehydration) -get A & B form depending on how much water take out of it, 1 twisted tighter than other; Z form twisted in opp direction

Watson & Crick DNA

-Nucleotide = Sugar + phosphate + Base -Single strands are anti-parallel in the double strand - complementary to each other. -The average 3D structure of a double strand is a double helix. -have major groove & minor groove (one just more exposed to base pairs); most proteins that bind to base pairs bind to major groove bc there's more space -10.5 base pairs/turn The Double Helix structure of DNA is a model (hypothesis) Q - How can we test the model? A - Dickerson Dodecamer (12 base pair piece of DNA)

Binding of O2 to Hemoglobin Depends on PO2

-O2 concentration alone isn't how we adjust structure & function (helps determine how/low affinity state); pH & CO2 also important -pH changes disrupt ionic bonds (same structural change as bending alpha helix) -in active muscles, low pH & want low affinity state (to release O2) -CO2 same as pH: high CO2 conc result in low pH (transport CO2 by combining w water -> form bicarbonate & proton) *can bend alpha helix by pulling up on iron, changing shape & physically separating ionic bonds *OR can reduce pH & break same bonds/have same shape change *shape change that occurs due to O2 binding is same shape change that occurs when lower pH; both result in breaking ionic bonds holding subunits together -in lungs w higher pH, in high affinity state *then go to low pH of muscles w low O2 conc & in low affinity state, will readily give up O2

Phosphodiester bonds

-Phosphodiester bond in the backbone of DNA links a phosphate group and the sugar (one bond to a 5' carbon and one to a 3' carbon). The phosphodiester bonds link successive nucleotide units. The backbone of alternating pentose & phosphate groups in both types of nucleic acid is highly polar.

Problem 1

-Problem - need a primer -Solution - make a primer (primase) *need primer to start DNA synthesis (simply provide DNA primer in PCR) *need primer bc DNA polymerases can't just find single-stranded template & start making new strand; can't make first base, need few bases already made to add onto -that's diff from RNA polymerases, which can *start* a new strand -Why is the primer RNA?: bc can't *start* new strand w DNA -problem bc made of RNA, later need to remove & replace w DNA (want chromosome to be all DNA) -primase = DNA-dependent RNA polymerase (start new strand, add couple 100 bases)

Problem 4

-Problem: twists created in DNA following unzipping -solution: gyrase (topoisomerase) - takes care of tangling of DNA *topoisomerase: enzymes that help control overall structure & organization of DNA -DNA replication in linear chromosomes slightly diff from circular chromosomes *both have leading & lagging strands, but circular chromosomes much smaller than eukaryotic chromosomes -DNA opened up, creating 2 replication forks moving in opp directions (each fork has leading & lagging strand) -once open up DNA (since double helix), as pulling/untwisting DNA, downstream of what opening up becomes w twisted & knotted -gyrase manages overall structure of DNA, can cleave 1/2 of double helix so other 1/2 free to rotate; unwind then seal up bind

Protein Folding

-Protein folding typically occurs as the protein comes off of the ribosome. It is *cooperative* (cooperativity of folding) process & is driven in part by hydrophobic effect to reach a low free energy conformation. Folding sometimes is assisted by other proteins (for example correcting disulfide bonds or exposing hydrophobic regions to the exterior part of the protein). -cooperativity means that folding of 1 part of protein not independent of the folding of another part of protein •The cooperative process is such that the initial formation of small elements of structure accelerate (direct/limit) subsequent structure development (folding). -once you form 1 stable structure in protein, directs how rest of protein can fold •In folding, the polypeptide chain goes from a high energy, high, entropy state (unstable) to a low energy, low entropy state (stable). (protein folding entropy driven) **energy stored in unfolded protein bc hydrophobic parts want to come together *as protein folds, entropy in universe increases even though protein gets more orderly; more energy when unfolded bc further from equilibrium -any hydrophobic regions have cage of organized water around; then clump together on inside of protein & liberate water; once more bonds in place, protein gets more stable

Nucleic Acids can form more than a double helix

-RNA can also form complementary base pairs (single-stranded messenger RNA typically doesn't fold into meaningful structures) -can fold into complicated 3D shape; can have weird base pairing (ex. uracil w guanine) **we can have Watson-Crick base pairing within RNA molecule; that's how we get complicated RNA structures like tRNA -Secondary structure of RNAs. Bulge, internal loop, & hairpin loop; additional complementary pairings add the the 3D structure -sometimes even non-Watson-Crick base pairing can occur

Ribonuclease A (enzyme this exp was orig done w)

-Ribonuclease A: 124 residue enzyme that breaks down RNA; it's a small protein -primary structure & 4 disulfide bonds (why so many?): their small size means they lack opportunity for a lot of other forces, so need extra stability of disulfide bond -How to denature this protein? (need reducing agent for disulfide bond & st else for the rest) *Urea: will denature all but disulfide bonds *β mercapto-ethanol (BME): a reducing agent, will break disulfide bonds -test is to remove denaturing agents & see if refolds properly; know if protein is functional if breaks down RNA into nucleotides; would do this slower (less activity) if slightly misfolded •What will happen when both denaturing agents are removed (via dialysis)?: protein would renature (& fold back) & restore function •What will happen if only one is removed? 1. Remove only BME: Reoxidation reforms -S-S but not necessarily in the correct place. *allow disulfide bond to form w/o other structures forming; get scrambled ribonuclease bc form wrong disulfide bonds 2. remove Urea (& include trace amounts of BME): *if want to take back to native structure, need to leave some BME (not enough that prevents reforming of new bonds); not only need right disulfide bonds to form, but also need to break incorrect ones *this is cooperativity of folding: allow this to refold w all molecular forces able to reform, then can direct correct disulfide bonds (need correct hydrogen bonds to form to form correct disulfide bonds) -the structure of one part of the protein (correct disulfide bonds) is not independent of folding of other parts of the protein (Cooperativity of folding)

Secondary Structure - review

-Secondary structure involves interactions between nearby amino acids in a peptide. Hydrogen bonds are very important in determining secondary structure. -bonds in alpha helix among first to form in folding protein bc hydrogen bonds every 4th residue, nearby bonds form first! -beta strands, depending on how distant interactions are, will also form relatively early/later on -R groups in alpha helix pointing out from axis; R groups in beta strand alternating

Types of Polymerases

-Taq (PCR): survive boiling; DNA-dependent DNA polymerase -Phage polymerase: phage=viruses that attack bacteria; DNA-dependent DNA polymerase -Reverse transcriptase: RNA-dependent DNA polymerase (use RNA as template to make DNA) *despite diff functions, work in same basic way: have template, read it in 1 direction, new strand synthesized in other direction (adding onto 3' end)

Fixing Telomeres

-Telomerase: RNA dependent DNA polymerase -person w/o telomerase age rapidly -telomerase = RNA-dependent DNA polymerase (reverse transcriptase); uses RNA template to make DNA -template only tiny fragment of RNA that comes w enzyme (polymerase carries its own template) -template binds to end of chromosome (bc complementary to that sequence) & add a few bases -telomerase finds ends of chromosome that's single-stranded; uses its RNA template to line up w end of chromosome & add new bases, make single-stranded piece on end of chromosome longer (extend chromosome) -once adds DNA, primase can come in, make primer, & we can replicate that -if sequence telomeres, sequence will be whatever's complementary to RNA fragment repeated over & over -some cells replicate a lot more than others, have telomerase activity more often; some rare telomerase activity -cancer: telomerase activity all the time

Review - Watson & Crick vs Dickerson's Dodecamer

-The structure of the 12 bp double helix differed from the Watson & Crick model predictions - it varied by sequence. Different sequences have slightly different conformations.

X Ray Crystallography (Rosalind Franklin)

-X-ray Crystallography is a method used to determine the arrangement of atoms of a crystalline solid in 3D space. -how we determine structure of large biomolecules incl proteins, DNA -take molecule; make it very pure, put it into v regular structure (arrange in regular pattern) -shine x-ray through molecule; if hits electron, x-ray defracted (look at defraction pattern, can determine structure)

What do proteins do?

-all of these bind to something; proteins do everything •Catalysis (enzymes) •Structural (collagen) •Contractile (muscle) •Transport (hemoglobin) •Storage (myoglobin) •Electron transport (cytochromes) •Hormones (insulin) •Growth factor (EGF) •DNA binding (histones) •Ribosomal proteins •Toxins and venoms (cholera & melittin) •Vision (opsins) •Immunoglobins (antibodies)

Prokaryotic vs eukaryotic initiation of replication

-bacteria have single origin of replication bc small circular chromosomes; 2 replication forks go in opp directions until meet -Prior to replication, specific proteins bind to DNA & begin unwinding at origin of replication. Other components of the replication fork are then recruited -Eukaryotic chromosomes (linear) contain multiple replication origins (within an AT-rich regions), where a helicase enzyme unwinds the double helix -euk need multiple bc chromosomes are larger, need more than 1 to replicate DNA quickly enough

Hershey-Chase Experiment (1952)

-bacteriophage attaches to bacteria, injects st into bacteria to infect it & then most of virus just detaches; part that's injected is enough to make new viral particles, so must be genetic material Method: •treatment 1 - radio-label phage protein (radioactive sulfur) •treatment II - radio-label phage DNA (radioactive phosphorus) •expose non-labelled bacteria to separately labelled phage (enough time for phage to inject bacteria) •remove phage (blender & centrifuge): use blender to separate bacteriophage from bacteria -what is in the bacteria? radioactive protein or radioactive DNA? •which labelled material was injected into the bacteria? -only saw radioactive bacteria in treatment II: meaning the material injected into bacteria was DNA not proteins; therefore conclude DNA is the material of hereditary Q: what's injected into cell when virus infects it? A: DNA

What to Know About Watson & Crick

-be able to describe basic details of their model & how they arrived at their model -hypothesis & observations used to make hypothesis: knew structure of nucleotides; Rosalind Franklin's x-ray crystallography data; & Chargaff's Rule (A=T,G=C) -part of their model that explains chargaff's rule: complementary base pairing (across double helix, A pairs w T, G pairs w C) -they described DNA as a double helix (2 halves held together by hydrogen bonds, twisted into helix); it's antiparallel (strands go in opp directions); hydrogen bonds hold base pairs together; H bonds weak, but there's a lot so DNA v stable; backbone alternating sugar & phosphate groups (why so soluble; phosphates neg charged on outside)

Mucins Summary

-be able to describe structure & say how that structure translates into the function •Mucins are large, extracellular glycoproteins with molecular weights ranging from 0.5 to 20 MDa. •Both membrane bound mucins, and secreted mucins share many common features. They are both highly glycosylated consisting of 80% carbohydrates (primarily N-acetylgalactosamine, N-acetylglu- cosamine, fucose, galactose, & sialic acid (N-acetylneu- raminic acid) and traces of mannose and sulfate). •The oligosaccharide chains consisting of 5-15 monomers, exhibit moderate branching and are attached to the protein core

PCR process

-begin w template for synthesis -have DNA, primers, taq polymerase, magnesium in tubes; put in thermal cycler -denature DNA, separate strands by heating: gets hot enough to break H bonds in DNA; if any proteins left in besides taq, they denature -lower temp so primers anneal to DNA (anneal=bind to what complementary to) -make temp that taq likes; if there's a single-stranded template of small region that's double-stranded, it will add bases onto 3' end of small fragment (copy DNA) -polymerases keep going as long as it has a template, beyond region want to copy -repeat those 3 temps, get exponential increase in DNA copies -even though regions outside of what defined by primers get copied early on; more cycles of replications, more you enrich only regions btwn primers

Beta Turn

-beta turn=4 amino acid residues within protein that make 180deg turn -secondary structure, so stabilized by nearby hydrogen bonds btwn nearby residues •β turns are 4 residue turns stabilized by hydrogen bonds btwn the first & fourth residues (have to make sure that 1 & 4 don't interfere w each other) •Proline or Glycine are usually found in a β turn, most likely (too flexible/rigid for a helix) -have covalent & non-covalent forces (hydrophobic effect, H bonds, ionic interactions); the more complicated a protein, the more non-covalent forces hold together 3D shape bc they need to change shape! always twitching *as gets more & more bonds, more likely to stay in structure if more stable than it was before *if bond not more stable, breaks & forms another one *cooperativity of folding: each bond/interaction in protein not independent of other interactions (ex alpha helix already in place, stable & can help direct folding of other parts of protein; alpha helix already in place restricts what other bonds can form) •what exactly does a structure tell us, and does not tell us about the function of a protein? *often see identical enzymes just glued together (dimer); having same subunit multiple times is additional way we can regulate activity of enzymes *every time you bind st new to a protein, change protein's structure (structure=function); so if have 12 copies of same enzyme bound together, binding st to 1 copy changes structure changes structure of others bound to it

Nucleic Acid Structure

-building block of DNA = nucleotides; need ribonucleotides/DNA nucleotides if want to synthesize new strand of RNA/DNA -Nucleotide = Sugar + phosphate + Base (we use 4 to make DNA; use a diff 4 to make RNA) -Nucleoside = Sugar + Base (no phosphate groups) -Nucleoside: bond between the sugar & base is a glycosidic bond -Carbons of the sugar are numbered 1' - 5' -The OH (hydroxyl group) in the sugar is the difference between DNA and RNA (if remove oxygen from C, it's deoxyribose for DNA; ribose for RNA) -bond btwn sugar & nitrogenous base = glycosidic bond

Chromosomes are made of both DNA & Proteins

-chromosomes=genetic material; made of both DNA & proteins •Which molecules (DNA/proteins) are the material of heredity? (debate bc proteins more complicated; DNA made up of 4 nucleotides, proteins made of 20 amino acids) •How to test hypothesis? -inject pathogenic bacteria into mouse (would usually kill mouse), normally encapsulated w protective polysaccharide coat -Capsular polysaccharides are the primary basis for the pathogenicity of the organism.

Denaturing curves for two different proteins (ribonuclease A & apomyoglobin)

-difficult to study protein folding bc happens quickly; easier to study how proteins denature -2 proteins denatured w temp (x-axis); how much denatured is y-axis -as add more temp, get more denatured -if no cooperativity of folding, could say every bond in protein independent (doesn't influence) another bond; if 100% cooperativity of folding, every bond in protein dependent on every other bond -know what curves look like (like S shape w less steep slope) -if no cooperativity of folding, would look p linear (just add more heat to break more bonds) -if 100% cooperativity of folding, would be straight vertical line (at 0, then go up to 100%, looks like step); if break single bond, whole protein falls apart • how are the curves different? • why are the curves different? -apomyoglobin shows less cooperativity of folding bc it's closer to a linear relationship (less steep slope); add more heat, have proportional effect on structure of protein -ribonuclease A: more cooperativity of folding; add more & more heat, there's a threshold to completely destroy; bonds broken around that temp were v important (slope steep)

DNA replication - where to begin?

-don't just pick random spot; there are distinct places where replication begins, called origin of replication -how find origin of replication on chromosome? *want as few Gs & Cs as possible bc of number of hydrogen bonds *includes a lot of As & Ts bc easier to unzip, less H bonds

Anti-freeze proteins Intro

-don't prevent freezing; prevent damage due to water freezing -water damages biology when freezes; solid water less dense, takes up more space; ice crystal expands & breaks cell -In solid water, each water molecule forms bonds with 4 other molecules. Frozen frogs have: •no heart beat, •no blood circulation, •no breathing, •no detectable brain activity •cannot move -all vital functions return within 1-2 hours when frogs thaw.

1 More Factor: Glycolysis

-glycolysis: break down glucose -do a lot of gylcolysis when doing a lot of respiration in active muscles (make a lot of 23BPG when breaking down sugar for energy) -23BPG is a byproduct of glycolysis, binds to hemoglobin & stabilizes low affinity state -muscles break down lots of glucose, so produce a lot of 23BPG; muscles need a lot of O2, so want muscles to have hemoglobin in T state (low affinity state) so release the O2 -muscles have high 23BPG conc; as hemoglobin moving into muscles, binds to center of hemoglobin **in muscles, want it to drop off O2; in lungs, want it to pick up O2

Chargaff's Rules (only really need to know #4)

-he broke down DNA into its 4 indiv nucleotides 1. The base composition of DNA varies from one species to another. 2. Different tissues of the same species have the same base composition. 3. The base composition of DNA in a given species does not change with an organisms age, nutritional state, or changing environment. Know this one: 4. In all cellular DNAs, the number of adenosine residues is equal to the number of thymidine residues *(that is, A=T)*, & the number of guanosine residues is equal to the number of cytidine residues *(G=C)*. The sum of the purine residues equals the sum of the pyrimidine residues; that is, *A+G (purines) = T+C (pyrimidines)* -In DNA, the total abundance of purines is equal to the total abundance of pyrimidines

(can't uncook an egg) BUT: All the information necessary to properly fold a protein is (usually) contained in its primary sequence

-heat a protein & denature it; let cool, it folds back to proper conformation bc no interference w primary structure; BUT can't uncook an egg -can't uncook an egg bc aggregate of denatured proteins far more stable than native structure (would take a lot of energy to sep proteins for them to fold indiv again) *explanation: hydrophobic effect disrupted; denaturing changes structure, exposes some of hydrophobic regions; if there's many proteins, these hydrophobic regions cluster together in massive clusters of proteins that are extremely stable *that's why denatured proteins don't readily renature -how do we know this? -Test of this hypothesis: fully denature a protein (don't disrupt primary sequence; disrupt secondary, tertiary, quaternary structures). let it renature. see if it reforms it native shape. ->protein should return to normal structure & have normal function once denaturing agent removed

Each Myoglobin or Hemoglobin polypeptide has a Heme ring

-heme ring has Iron atom in center, held by 4 N in ring (to provide electrons if it loses any) -at bottom, held in place by histidine (contains N, so really it's bound by 5 N) -can form 6 covalent bonds: 5 held by N, so can make or break to Oxygen -atypical of protein-ligand interactions bc usually non-covalent (this is covalent) **when O2 binds, pulls up on Iron & changes shape of protein (allosteric) • Iron (Fe2+) is at the center the Heme prosthetic group. • Iron is bound by Nitrogen (x4) to the larger porphyrin ring. • Histidine binds iron from the bottom. • When O2 is bound, additional hydrogen bonds are formed with upper Histidine. •can also bind CO! :(

Myoglobin and Hemoglobin differ in the quaternary structure

-hemo & myoglobin have v similar tertiary structures -hemoglobin also has quaternary structure; myoglobin has single polypeptide, hemoglobin has 4 (tetramer/dimer of dimers) -both have heme (porphorin ring) • Closely related, and similar shape and function. • Myoglobin has 1 subunit (1 polypeptide) • Hemoglobin has 4 subunits (2 alpha, 2 beta) -Myoglobin is an O2 storage protein -Hemoglobin is an O2 transport protein

Hemoglobin, O2 and CO2

-hemoglobin found in red blood cells, transports O2 (pick up from lungs & drop off elsewhere) -myoglobin in muscles, emergency backup O2 release (gives couple extra seconds of activity) -pH? -when exercising, muscles have lower pH than lungs; pH reduced in tissues more active in terms of respiration bc of what we do w CO2 -CO2 nonpolar, can't dissolve in blood to transport it; take carbonic anhydrase (enzyme) which combines CO2 & water to make bicarbonate & proton *every molecule of CO2 we have to transport out of muscles, we produce 1 proton; this reduces pH by 1 proton (more exercise, more CO2 make, lower pH of tissues) • Some CO2 dissolves in the blood - but very little • Some CO2 binds to hemoglobin - any peptide amino end • Most CO2 is converted into HCO3- *one H+ produced for each CO2/reversed in lungs -that's why pH in active muscles lower than when not working out & lower than lungs

A Peptide

-how many possible interactions (contact points) are there within one peptide? -hydrophobic regions? hydrophilic regions? every protein has both -the big question - how does a peptide fold into a protein? -All the information needed to fold a protein is (usually) found in the primary sequence - but how does it 'know' how to fold *goes from extended polypeptide to v specific, distinct globular shape *proteins in 3D shape held together in compact shape by hydrophobic interactions, caused it to collapse in water; can then be other ionic, hydrogen bonds *hydrophobic collapse causes it to be compact *how can other regions interact? ends have pos amino group & neg carboxyl group; lots of combos for ionic & hydrogen bonds w R groups

Hydrophobic interactions help proteins fold

-hydrophobic core (with mostly non-polar R groups) -the outside is mostly hydrophilic groups - which can form hydrogen bonds with water *every protein doesn't fold in same way, adjust until all other bonds that stabilize protein come into shape (although all compact w hydrophobic center)

Polymerases & some orgo (start L 10/1)

-incoming NTP attacked at the alpha phosphate by the 3' neg hydroxyl of the growing DNA chain -> causes release of 2 phosphates & forms bond -this is the polymerase rxn we talked about last time -getting rid of a product of rxn is often how we can make rxn more likely to occur

Mucins (present in all animals)

-keeps mucous slippery (mucous stops infection, barrier btwn you & outside world) -mRNA sequence & AA sequence (primary structure): same thing repeating -glycoprotein: long protein that doesn't fold up into compact shape w lots of sugars attached; peptide backbone (CCN), repeated structure, chains of sugars (oligosaccharides) *function of all the sugar: soluble (hydrophilic) & take up lots of water & space -cohesiveness of mucous: interaction of sugars w water & w each other -forms gel-like structure (fibers bound together)

Transmembrane vs Secreted Mucins

-mucins stay attached to cells supposed to protect -attach alpha helix long enough to go through mem w only hydrophobic R groups to end of mucin; alpha helix embed in cell mem w mucin part sticking out -transmembrane mucin: ones w alpha helix that embeds in membrane; stick to secreted mucin (secreted from cell) -mucin concentration decreased farther from cell

O2 and CO2 do not interact well with H2O. why?

-need hemoglobin & myoglobin bc O2 & CO2 are nonpolar, can't dissolve in blood/interact w water -so need to bind them to st that can interact w water (protein) -Life requires Oxygen - which is a problem for us large multicellular organisms, because O2 and CO2 are not very soluble water

Back to Nucleotides

-nucleotides: how we build RNA & DNA, how we get sugar-*phosphate* backbone -which are needed to make DNA? which are in DNA? (diff versions based on # phosphates attached) *ATP, GTP...: 3 phosphates (vs mono/diphosphate) -how many phosphates found in DNA per nucleotide?: 1 (backbone=sugar-phosphate-sugar-phosphate...) -to make DNA: even though only 1 of those phosphates ends up in DNA, we need the triphosphate version; start w NTP & part of reaction of adding new base is losing 2 of the phosphate groups (that's the energy that drives that rxn) *only monophosphate found in DNA, but need triphosphate version to build RNA/DNA

DNA Replication

-only need 1/2 of double helix to make other 1/2; if know sequence of 1 half, then know sequence of other -Watson & Crick said to replicate DNA: unzip it & use 1 half as template to make other 1/2 for both strands -The preexisting or "parent" strands become separated, and each is the template for biosynthesis of a complementary "daughter" strand -structure of DNA immediately told us how it replicates & functions -Another prediction of the Watson-Crick Model: the replication fork

Polymerases

-polymerase: proteins that synthesize RNA & DNA •Enzymes that synthesize DNA in a *template dependent manner* *template=need strand to make new strand (need to copy st) •Can make DNA or RNA complimentary to the template strand •Template can be DNA or RNA -ex: if polymerase copies DNA, "DNA-dependent DNA polymerase" (reading DNA to make DNA) - have diff templates & diff products •Accuracy can be very high (can be less than 1 mistake in 1 mil bases) •Many can edit - detect and fix mistakes •Most organisms have several different types

Problem 5

-problem: no 3' to 5' polymerase -solution: leading & lagging strands (lagging strand made in pieces) -Because both daughter DNA strands are polymerized in the 5′-to-3′ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments -leading strand: start w primer, keep adding onto 3' end of growing strand until run out of template -lagging strand: as helicase moves in direction & opens up DNA more, polymerase on this strand can't move in this direction (bc can only add onto 3' end, DNA antiparallel) -so start w primer, build off in that direction; helicase has opened up DNA more, so need to back up & put another primer there; keep moving that direction until hit first piece; repeats *working backwards in small steps on lagging strand bc polymerases can only add onto 3' end

Protein Binding

-protein binds to ligand (could be anything) -Ligand - A molecule that binds reversibly to a protein. A ligand may be any type of molecule, including another protein. •Ligands bind to specific sites on the protein (ligand binding site) •The ligand binding site usually exhibits molecular complementary to the ligand in terms of: *size *shape *charge (pos & neg charge) *hydrophobic or hydrophilic characters (hydrophobic w hydrophobic; hydrophilic w hydrophilic) -characteristics should be opposite so can bind •Protein-ligand binding is very specific. • Binding of ligand to a protein will usually change the shape of a protein (sometimes very minor, sometimes major)

Specificity of binding is due to molecular complementarity

-protein-ligand interactions can be simple or complicated (v specific w high affinity) -want to make active site that's binding ligand to be as complicated as possible & be complementary to it •protein-protein interactions •protein-ligand interactions •enzyme-substrate interactions

Quaternary Structure

-same as tertiary structure, but has more than 1 polypeptide -non-covalent forces + disulfide bonds • Different subunits (usually) held together by non-covalent forces. -The 3D structure of a multi-subunit protein - particularly the way subunits fit together. -Many enzymes exist in large complexes of repeated units. why? *for purposes of regulation; every time you bind st new to a protein, you change the protein's structure (structure=function); so if have 12 copies of same enzyme bound together, binding st to 1 copy changes structure of that copy & changes structure of others bound to it

problem - enzyme falls off

-solution - clamp protein -in in vivo, polymerases replicate a few bases then fall off -sliding clamp binds to polymerase & hugs DNA so it doesn't fall off

problem - gaps between fragments on lagging strand

-solution - ligase -build DNA w Okazaki fragments: on lagging strand, put down primer then DNA; then put down another RNA primer & add DNA -RNA then gets removed & replaced w DNA by another polymerase -but then have gaps in backbone bc built in fragments; there's a missing bond bc built lagging strand in pieces -have another enzyme come in (ligase), uses energy from ATP & completes that final phosphodiester bond in backbone

problem - leading strand faster than lagging strand

-solution: polymerase dimerization (2 polymerases bound together) -lagging strand more complicated, takes longer to replicate; don't want leading strand replicated long before lagging strand; need to coordinate replication of both strands -polymerase dimerization: simple protein binds to 2 copies of polymerase, so 1 can't get too far ahead of the other

DNA vs RNA

-structurally almost same, but functionally v diff 1. structural differences: -RNA usually not uniform double helix (though can often be double stranded); ribose vs deoxyribose; instead of thymine in RNA, have uracil -Why use use T in DNA rather than U? *U & C similar, so mistake in U read as C. So have T in DNA bc not structurally similar *but then: why both RNA & DNA, why not just 1 or the other? 2. Functional differences: -make RNA from DNA, used to transfer info from DNA to make protein (make protein using RNA intermediate) -ribosomes made of RNA + proteins (peptide bond catalzyed in ribosome by RNA) *RNA world hypothesis: before there was DNA, maybe there was RNA; organisms had genomes made by RNA (& RNA can fold up into complicated structures & act as enzymes) *so maybe before proteins & DNA, there was just RNA (so it was genetic material + catalyst)

Polymerases (start L 9/28)

-synthesis is 5' to 3' (we add onto the 3' end of a growing strand) -every polymerase *adds onto the 3' end of a growing strand* (5' to 3' synthesis), reading the template strand in the opposite direction (3' to 5') -Many DNA polymerases (not all polymerases) have a *polymerase* function (synthesis of new complementary DNA strand) as well as editing function (*exonuclease*, removal of incorrect bases) *polymerase part adds new bases; exonuclease removes bases (digests nucleic acids) -polymerase determines next base by whatever's complementary to template strand -if make a mistake, changes structure of double helix (no longer correct), which changes structure of protein; this moves newly synthesized region down to exonuclease region, which chops off a few bases until have proper double helix *then shifts back up to polymerase region & continue adding bases -some of our DNA polymerases can spot mistakes when they occur & correct them immediately *some mistakes can't be caught; polymerases specialized in finding & editing mistakes correct these mistakes *we have multiple opportunities to fix mutations in DNA -Most polymerases share a conserved overall shape (incl fingers, thumb & palm regions), suggesting ancient common origin *palm=active site, where DNA sits; moves through palm as synthesizing new DNA

What is a gene?

-the functional unit of our DNA; contain info used to make RNA -usually code for a trait/protein -some genes never have their info make it to protein (like tRNA, its sequence isn't used to make peptide) -all genes code for some form of RNA -have 20-22k genes; make 100k proteins -have coding region (code for RNA that might become protein) & regulatory region

Post translational modifications

-those carbs are an example of post translational modification (ribosome didn't put them on, another enzyme added them after polypeptide made) -post translational modifications done often in ER & golgi, some of these mucins made there •Why would these proteins be made in the Rough ER and not on free ribosomes? *free ribosomes produce proteins used by cell; attached ribosomes make proteins transported out of cell -Extensive post translational modifications!

tm is the half-way melting point

-tm=amount of heat to unzip 1/2 DNA (denature 1/2 bases) -each DNA molecule has a different tm. why? -The Higher the GC content, the higher the tm. WHY? **there's 1 more bond btwn G & C than btwn A & T; talking about GC content **base composition of DNA determines how many H bonds holding it together, which determines how much heat it takes to unzip DNA -the more Gs & Cs in genome, the more stable it is to being denatured by heat (linear relationship btwn tm & GC content) **extremophiles have lots of Gs & Cs in genomes; we have regions that are more enriched & others heavy in As & Ts **why some regions easier to unzip than others in us?: when replicate chromosomes, have to first unzip them; place where we unzip enriched in A & T bc easier to unzip (get partially denatured DNA) -another functional consequence: when express our genes, have to unzip DNA v locally & make RNA copy (unzip them in place enriched in As & Ts)

Disulfide Bond Formation

-to break disulfide bond, need to add electrons (redox reactions, need reducing agent to break bonds)

Transcription Intro

-transcription: taking DNA template & making RNA (we talk about more specifically mRNA) -Highly expressed genes may produce multiple RNA copies at the same time (by multiple RNA polymerases) -RNA polymerase just rides along chromosome, reading 1/2 of double helix & making complementary RNA; longer it goes along the chromosome, longer the tail of RNA that's coming off -in prokaryote, can have transcription & translation at same time: ribosome jumps onto RNA & start making protein before bacteria finishing RNA *euk: RNA has to go out of nucleus into cytoplasm -how does polymerase know where to begin & end?

Lagging vs Leading Strand

-unzip DNA & have 2 single strands, one leading & one lagging (need to distinguish them bc only work in 1 direction) -synthesize new strand 5' to 3'; read template 3' to 5' -Leading Strand: just start replication & polymerase just keeps going until hits end of chromosome -Lagging Strand: not so simple

PCR (polymerase chain reaction)

-wanted way to copy short pieces of DNA -goal: copy v specific piece of DNA -Ingredients: *DNA template (something to copy) *Polymerase (enzyme to do synthesis) *primers: short pieces of single-stranded DNA (length need to be unique); want primers to bind to unique sequences in DNA; primers define where you're replicating *Nucleotides (NTPs): what we build DNA out of (need triphosphate version, NTP) *also Mg+ & buffer

Denaturing

-we can easily denature DNA like we can proteins; just have to disrupt H bonds (easier than proteins) -DNA can be unzipped by breaking hydrogen bonds - how? (water not enough) HEAT!! -Reversible denaturation and annealing (renaturation) of DNA *heating hasn't destroyed single strand; unlike proteins, if cool DNA down it will easily zip back up

DNA synthesis catalyzed by DNA polymerase (end L)

-we have specific polymerases for making tRNAs, ribosomal RNAs, messenger RNAs; as well as copying vs editing -every polymerase has the same basic structure (looks like hand holding onto piece of DNA; active site in "palm" holding onto DNA) -some polymerases are 2 enzymes in 1 a. can polymerize (that's the rxn that adds new bases; read template & adding new base that's complementary to whatever base is in template) b. another region removes bases (opposite function): if make mistake (wrong base added, messes up shape of double helix); proteins change conformation when bound to something diff; result=end of growing strand moved to diff region in polymerase *shift mistake region down to proof-reading portion of polymerase & it backs up, chops off bases until double helix has right structure; then goes back up to polymerizing region & adds more bases (some polymerases can catch mistakes as occurring; back up & remove mistaken bases, then continue) -Kornberg's polymerase: after replicate DNA, looks around for mismatches & removes bases, then adds correct one

Low pH

-when/where is pH low? -these proteins are largely unfolded, but have small regions folded into hydrophobic globular -expose those by denaturing protein by lowering pH a little (like w infection), then hydrophobic effect can cause more than 1 of those regions to bind together

Types of Proteins (don't have to memorize all types, just an overview)

1. Outside of Cell -tend to be small if outside of cell -hormones (send messages to & from cells), digestive enzymes (break down proteins), antibody 2. Membranes (some allow thing in/out of cell; some make ATP) 3. Transport & Storage (transport across cell mem/throughout body) 4. The chemists (enzymes) 5. DNA (many proteins interact w DNA) 6. New protein synthesis (or modifying proteins) -ribosome: made of RNA & proteins; enzyme that forms peptide bonds; region that forms peptide bond strangely only RNA 7. Structure & movement (cytoskeletal proteins): can be cytoskeleton or like collagen, that literally makes up our skeleton -collagen, actin, microtubule

Structures

1. Primary structure: sequence of amino acids within a peptide (covalent bonds) 2. Secondary structure: stable conformations of the peptide chain from rotation interactions between nearby AAs. e.g. α-helices and β-sheets, β-turns (local interactions, hydrogen bonds) -can have beta turn connecting beta strands, but no need; can have beta turns connected to other things & have beta strands connected to other turns 3. Tertiary structure: 3D shape of the fully folded polypeptide chain (3D structure of single polypeptide folded into native configuration); held together by 4 non-covalent forces + covalent disulfide bonds -small protein has fewer opportunities for other bonds to hold together, so need extra stability from disulfide bonds (have more cysteine) 4. Quaternary structure: 3D coordinates of everything in structure w more than 1 polypeptide

Problems 2 & 3

1. Problem: No single stranded template -Solution: Helicase unzips DNA 2. Problem: single strand stability (single strands are unstable...don't stay single for long) -Solution: single strand binding protein -once DNA unzipped, wants to zip back up if not kept at high temp -we have single-stranded binding proteins that bind & stabilize single strands of DNA long enough so they can be replicated *bind after helicase unzips DNA

How does in vitro PCR differ from in vivo DNA replication?

1. example: denaturing, separating 2 strands to replicate DNA -PCR: heat DNA strands to high temp to break H bonds -in vivo: have enzyme to unzip DNA

Conclusions from this Experiment

1. primary structure contains info necessary for tertiary & quaternary structure *as long as don't disrupt primary structure (sequence of amino acids), then have info necessary for protein to fold *should fold back into native structure when remove denaturing agent if let fold back in isolation (not interacting w other denatured proteins) 2. folding of 1 part of protein not independent from another part of protein

Graphs

1. sigmoidal bc hemoglobin not 1 protein, it goes back & forth btwn high & low affinity states -determined by CO2, pH, O2 conc (which is ligand conc) -it's a protein that changes shape w ligand conc; shape changes function -have 1 version that really wants to stick to O2, 1 version wants to give up O2 *in lungs, those factors contribute to picking up O2 v efficiently *in muscles, low pH breaks ionic bonds & low conc of O2 makes it easier to give up O2 -> both of these contribute to releasing O2 in muscles, picking it up in lungs 2. myoglobin more "sticky", has higher affinity than hemoglobin; gets its O2 from hemoglobin *if myoglobin didn't have greater affinity, wouldn't be able to pick up O2 from hemoglobin; also means doesn't release it easily, makes sense bc only released after deplete O2 from hemoglobin 3. human fetal hemoglobin has higher affinity: have completely diff set of genes to make hemoglobin when fetus bc get O2 from mother's adult hemoglobin

Dickerson Dodecamer

Method: •Create a molecule of known sequence (12 base pairs, double stranded; know where every bond is) *only had to synthesize 1 molecule bc its complementary to itself; let it bind to itself •Analyze the structure via X-ray crystallography ->is this really a double helix? Results: •Differs from the perfect Watson & Crick helix in several minor ways: -some bases in the dodecamer were twisted slightly - not a feature of the Watson & Crick model -The slight departure from the 'perfect' Watson & Crick model is due to sequence differences **bent slightly; this piece of DNA bent 19deg (but not all DNA) **bond angles twisted; base pair more twisted; not perfectly smooth like Watson & Crick **a lot of variations in bond angles

Proteins fold into more stable structures (end L1)

Molten Globule model of protein folding - • Hydrophobic interactions cause protein to first form a 'molten globule' (not the final form). • Future folding brings the protein into its final shape. • Multiple paths to the final protein. • *Thermodynamics drives protein folding* • *Entropy decreases with protein folding* *"folding funnel": explains entropy-driven process of protein folding; funnel shape nature means that although 1 destination, there's many ways to start folding *top represents unfolded -> bottom=native structure (properly folded version of protein, most stable) -Under appropriate refolding conditions, the molecule condenses around a hydrophobic core into a compact, but non-native, intermediate, called a molten globule. In this folding intermediate, much of the secondary structure is present. Long-range interactions then form the tertiary structure, folding the molecule into its native 3D conformation. *"self-solving puzzle": hydrophobic collapse & formation of secondary structures *secondary structures form first, nearby interactions; then distant interactions can form

Protein-Ligand Binding

P + L --><-- PL (bind if mixed together) -the more complementary, the better the binding -protein concentration kept constant; each tube has increasing ligand concentration -as add more ligand, bonding more -lower the Kd (substrate conc that binds half of proteins), better binding •The proportion of protein bound to ligand will increase as the ligand concentration increases (if talking about low ligand conc) -at some point, stops increasing bc saturated proteins •The % protein bound will usually never be 100% (why?) -levels off, doesn't reach 100% bc forces that bind proteins & ligands are non-covalent so not permanent (some unbind, bind back together) •Each protein-ligand interaction will have its own characteristic binding curve. •Kd is simply a useful way to compare different protein-ligand interactions (how much ligand is necessary to make the protein binding sites 'half full'). Smaller Kd = better at binding

The problems with in vivo chromosome replication (not seen with in vitro PCR)

Problem -> Solution (different proteins) •No single stranded template -> Helicase (separates DNA strands, bc cells can't just boil themselves to get single-stranded template) •The single stranded template is not stable -> Single Strand Binding Protein (RPA) •No Primer -> Primase (RNA bc can't start new strand w DNA) •no 3' - 5' polymerase -> Replication fork (leading & lagging) •Enzyme falls off template -> SSB & sliding clamp •leading strand is much faster -> Polymerase for both strands is a dimer •multiple fragments on lagging strand -> DNA ligase •twists created in DNA following unzipping -> Gyrase ...so things are more complicated in cells than in PCR tubes

Arthur Kornberg (1959) - discovered first polymerase

Took a biochemists approach (diff from geneticist) - wanted to find polymerase, protein that's responsible for copying DNA: • grind up E. coli (take organism of interest & grind it up) • separate components (sep proteins into as many diff fractions as you can) *sep into 1000 diff tubes, each has diff combo of proteins; of those 1000 tubes, 1 of them at least has protein that copies DNA • look for polymerase activity in different components (added template, primer, radio-labelled nucleotides...looked for new strand synthesis) *go through tubes to see what makes DNA; need an assay (provide nucleotides & see if enzymes in that tube have synthesized new piece of DNA) • found a component with polymerase activity (DNA polymerase I) • win nobel prize

Types of RNA

• *Messenger RNA (mRNA): codes for polypeptide • *Transfer RNA (tRNA): brings amino acids to mRNA • *Ribosomal RNA (rRNA): componenets of ribosomes • Small nuclear RNAs (snRNAs): part of ribonucleoprotein complexes • MicroRNAs (miRNAs): function in gene regulation • Small interfering RNAs (siRNAs): help defend against genomic parasites * = most common *don't need to memorize, just appreciate in addition to 3 major classes, there's lots of other types of RNA (many inv in regulating gene expression) -of common types of RNA, ribosomal most abundant ~ 80% (then transfer, then messenger only ~ 5%)

Dickerson Dodecamer Conclusions

• 3D structure of DNA varies with sequence! (main conclusion) -Watson & Crick were correct, but they proposed the *average structure of DNA* based on an entire genome (bc based on Rosalind Franklin's x-ray crystallography) -Dickerson: if look at exact structure of any piece of DNA along chromosome, going to be basically what Watson & Crick described, BUT specific details of double helix different based on sequence • Differences in twist, roll, propeller twists, displacement...(differences in many minor features) • Departures from B DNA by sequence -why is this so important?: every sequence having slightly diff structure in terms of double helix means that every DNA sequence is a diff ligand!! **if cell wants to locate v specific sequence present only in 1 specific gene, have protein that will bind only to that sequence; only possible bc structure of DNA varies by sequence -why imp: proteins bind to DNA in sequence specific way; if every sequence had same structure, wouldn't be able to have proteins that bind to specific sequence; *binding proteins to DNA in sequence-specific way is how we regulate gene expression!!*

Tertiary Structure

• The 3D folding of a polypeptide is its tertiary structure. • Both the α-helix & β-sheet may exist within the tertiary structure. • Generally the distribution of amino acid R groups in a globular protein finds mostly nonpolar residues in the interior of the protein and polar residues on the surface. • Tertiary structure is maintained by noncovalent interactions and disulfide bonding. -Tertiary structure refers to the relative locations in 3D space of all the atoms in the molecule. The overall shape.

How is the information carried in DNA manifest?

• The DNA structure varies by sequence (and time). • Proteins recognize variance in DNA structure (according to sequence) -lesson of dickerson dodecamer: we can read sequences of DNA bc of their structure

Rough versus Smooth strains of Streptococcus pneumonia bacteria? (Avery exp)

• named for their appearance when grown as a colony on a petri dish. R strain was derived from S strain. • colonies have different morphology due to inability to form a polysaccharide capsule in the R strain • S (smooth) strain is DEADLY to mice • R (rough) strain is NOT DEADLY to mice *R variant of same bacteria doesn't have polysaccharide coat & can't kill mice -if boil S strain, doesn't kill mouse when injected -if boil S strain, let cool, then mix w R strain (non-virulent): kills mouse! • Information necessary for causing virulence survived the heat treatment of the S strand and was transferred to the R strain. *R (non deadly) was 'transformed' into S (deadly).* • What molecule(s) contains this 'virulent' information? A: DNA! (material of hereditary passed to non-virulent strain) • How to test? -repeat this experiment, but remove 1 type of molecule (DNA or proteins) before mix dead & live bacteria *removing proteins killed mouse (so proteins not imp for killing mouse); removing DNA does not kill mouse *whatever info transferred from dead to live bacteria must have been found in DNA

DNA Polymerase I (what he found)

• works only in 5'-3' direction (can only add new bases onto 3' end) *every polymerase works in only this same direction • cannot start replication from single stranded DNA (can only add onto pre-existing strand) • requires ATP, CTP, GTP, TTP (why not AMP...?) *requires NTP version (3 phosphates on each nucleotide, bc lose 2 phosphates as energy to make new bond) • needs primer: something to start out strand • requires magnesium (positively charged, stabilizes neg charges in phosphate groups) • the enzyme falls off after only a few bases (copy maybe couple hundred bases then stop)

Early Milestones in Genetics

•1865 - *Gregor Mendel* provides first evidence for a hereditary material (and begins Genetics) •1910- *T.H. Morgan* provides evidence that *chromosomes* are the material of heredity *Two Classic experiments established that DNA (not proteins) is the material of heredity* •1928-1944 - *Transformation experiments* with Mice & Streptococcus pneumonia bacteria. (Griffiths, then Avery et al.) •1952 - an experiment with *Bacteria & Bacteriophage (Hershey-Chase* experiment) •1953 - *Watson & Crick*, DNA structure

Structure of Antifreeze Proteins - how bind to ice

•Antifreeze proteins bind to ice •Beta helical structure w evenly spaced hydroxyl (OH) groups on the flat side allows for H bonds with water crystal •This makes it thermodynamically unfavorable for water molecules to add to the ice lattice. •Resulting ice crystals remain the same size for hours or days at freezing temperatures. *protein mimicking ice (similar surfaces); form H bonds & prevent growth on surface of ice *hydrogen bonds within protein holding adj beta strands together

Hemoglobin Summary

•Binds, transports and releases O2 (unlike myoglobin) •O2 binds heme. CO2 does not •Has two different conformations (R & T states) •Positive, allosteric binding cooperatively helps hemoglobin release oxygen in tissues and bind it in the lungs. •pH influences O2 binding - protonation of key residues stabilizes the T state •CO2 transport (binding to Hemoglobin Amino end, or conversion to bicarbonate) lowers pH. *be able to describe transition of hemoglobin from lungs to muscles & back again *moving from muscles, just dropped off all O2 -> heart (veins, arteries) -> lungs (have higher pH, fewer free protons), ionic bonds more stable, won't be broken & much higher O2 conc *when first no O2 bound & first moving into lungs, in low affinity state (bc no O2) *as move from low pH of muscles to higher pH, higher O2 conc (lower CO2 conc) - all these contribute to stabilizing high affinity state *in low affinity state until bind first O2 -> changes shape of everything -> now can pick up 3 more O2 -> now in high affinity state -> go back to muscles, pH lower -> protons bind to neg charged regions of hemoglobin, making ionic bonds impossible (break ionic bonds that stabilize state we were just in) -> release O2, changing structure of subunit & other subunits -> further biasing back to low affinity state -pH & O2 conc changing in tissue of lungs & muscles is what converts protein back & forth to high/low affinity states *high affinity in lungs to pick up more O2; low affinity state in muscles to drop off O2

Watson & Crick Model Summary

•DNA is a double helix (goes around central axis, held together by H bonds) •Anti-parallel (2 halves of double helix run in opp directions) •Hydrogen bonded base pairs on the inside -only specific base pairs bond together (A binds to T, G binds to C; come in pairs) *can have any combination of sequence *A & G, T & C have similar structures -A & T held together by 2 hydrogen bonds; G & C have 3 hydrogen bonds •Sugar-phosphate backbone on the outside (soluble bc phosphates neg charged on outside) •Each chain runs 5' to 3' •Implications of model: complementary strands suggest mechanisms of replication *if know sequence of 1/2 of double helix, then you know the other 1/2; this immediately suggested how DNA copies itself (know just from structure)

Know the function of the following in DNA replication: (end L 9/28)

•Helicase •Single Strand Binding Protein (RPA) •Primase •Replication fork (leading & lagging) •SSB and sliding clamp •Polymerase for both strands is a dimer •DNA ligase •Gyrase •telomerase (RNA-dependent DNA polymerase)

Some More Questions to Ponder (end L)

•How would O2/CO2 transport differ if hemoglobin was a monomer? dimer? •How would O2/CO2 transport differ if red blood cells had myoglobin instead of hemoglobin? •How exactly does CO2 affect pH? •Why hasn't hemoglobin evolved ways to deal with CO?

Rosalind Franklin

•The X shape suggests a helix (from x-ray crystallography) •10 lines suggests 10 units/turn & 34 Å/turn •Described 2 different forms - dependent on water content •Phosphates lie on the outside, sugar & bases inward -James Watson was shown Franklin's data without her knowledge. Without her work, Watson and Crick would never have made their model

Some proteins contain chemical groups other than amino acids

•The non-amino acid part of a protein is usually called a prosthetic group. •a cofactor is an inorganic ion or a coenzyme required for enzyme activity. •a coenzyme is an organic cofactor required for the action of certain enzymes, often has a vitamin component. -prosthetic groups: parts of proteins/enzymes necessary for rxn but not made out of amino acids -Non-amino acid materials can be very important for protein structure/function: example= calmodulin inactive & have diff structure w/o calcium bound to it; calcium changes shape & makes it functional

Antifreeze Proteins

•The problem with water: when water begins to freeze, many small crystals form, but then a few large crystals dominate & grow larger & larger, stealing water molecules from the surrounding small crystals...and may rupture a cell •Antifreeze proteins counteract this recrystallization effect (small crystals getting big), making it thermodynamically unfavorable to add more water to the ice •Antifreeze proteins bind to the surface of the small ice crystals and slow or prevent the growth into larger dangerous crystals. -Flat, hydrophilic (threonine-rich) face of molecule adsorbs to surface of ice crystal. -Hydrophobic face repels liquid water molecules, inhibiting further crystal growth. *hydrophilic size flat, regular; hydrophobic side not regular *flat side bind to surface of ice *prevent new water molecules from being added near protein

Examples of Ligands

•substrates (ligand for enzyme) •signaling molecules (send info to cell; like insulin) •antigens •inhibitor drugs (a lot of drugs we used simply inhibit enzymes)

Beta Sheets

•β strands tend to not run straight, but take on a slight twist •where are the R groups? every other one going in opposite direction -can put a lot of beta strands together & make beta barrel: can be a pore (like porin), spans a membrane 1. can make pore w barrel *Q: think about R groups in primary sequence of 1 strand within beta barrel; if want to design protein so embedded in membrane & inside of it is hydrophilic (so hydrophilic things can move in/out of mem), how would you design the primary sequence of protein? *A: it should alternate hydrophilic, hydrophobic, hydrophilic, etc...; so those pointing outward is hydrophobic & inside hydrophilic 2. can make GFP (fluoresces, from jellyfish & can put in other organisms) with barrel *in barrel bc the part that fluoresces on inside needs to be protected *put GFP in mice by adjusting genes (take gene from jellyfish to mouse); "reporter gene"; take gene that you don't know when/where it's expressed & add GFP, make GFP anytime that gene should've been expressed