Lecture 1 Read the material before AND after lecture 6th edition: 20-30 $ on Amazon TSA office hours:

hours: Tuesday 10-11 in 3340 medical science research building III Instructor office hours: Thursday afternoons Get a three ring binder and print out notes Use flash cards for memorization

What is Biochemistry? Conversion of 6 C glucose that is oxidized by oxygen to give co2 and h20 allows life to grow and thrive What reactions catalyze this reaction? And what are the energy intermediates?

Chapter 1 Chemical Bonds Not all bonds and interactions are of the same strength Chemical bonds (strongest): covalent bonds o Electrons share e-, double bonds stronger than single, enzymes lower AE to break bonds during reactions Electrostatic interactions o Interactions between ions, defined by coulombs law o Dielectric constant (D): accounts for home much electrostatic screening there is high D, water (polar) has dipoles that align themselves with dissolved ions and neutralizes reaction H bonds o Formed between H donors (EN atoms bonded to H) and H acceptors (EN atom with no H, free pair e-) o Bond angle and distance are important: ideal angle is linear Water is asymetrical each water can accept 2 H bonds and donate 2 H bonds (2 lone pair e- on Oxygen) form a cohesive network (lattice structure forms in ice) Van der waal interactions (weakest) o All atoms have e- clouds, partial dipoles form due to momentary fluctuations of e- around atoms Hexane: dont dissolve because molecules stick together (CPK model) Each atom has an ideal distance van der waal radius with which it can interact with neighbouring atoms tables, experimentally measured Water forms cage around non-polar molecules reduce entropy (energetic penalty) in water molecules that participate in this cage

Lecture 2 Refresher: talked about interactions and bonds these are the stabilizing forces Na and Cl ions interact with energy of -1.4 kcal/mol (negative # is attractive interaction) opposite charges attract Van der waals: fluctuation in e- clouds of atoms create temporary dipoles causes attractions (weak interactions between atoms) there is an optimal distance for this interaction (optimal attraction) called the VAN DER WAAL CONTACT DISTANCE Van der waals are the weakest interactions but the most numerous since charges are separated between atoms, the attraction is weaker (coloumbs law) when e- clouds overlap, there is repulsion at close distance (look at plotted graph) Water can form up to 4 H bonds: donate/accept 2 H bonds forms a structure of lattice (ice)

Stabilizing forces are now applied to DNA structure DNA is found in all forms of life uses chemical interactions to function Structure of DNA: deoxyribose nucleic acid encodes genomic information in cells (primary function) Structure: linear polymer with 4 monomers Double helix: 2 strands of DNA wound around each other anti parallel orientation One DNA strand: monomer (units called nucleosides) phosphor diesther bonds link monomers together P has charge Bonded to sugars is a nitrogenous base via glycosidic bond purine (have 6 and 5 membered ring) and pyrimidine (6 ringed) differences in chemical structures allows each to H bond differently Purine + pyrimidine H bond together between N and H, H and O GC 3 H bond, AT 2 H bonds H bonded nucleosides stack on top of each other inside the double helix bases tend to be hydrophobic (which is why they are inside the DNA structure) water is absent INSIDE DNA How does double Helix form? Hybridization of monomers Key structural element: base structure complimentarity DNA replicates before mitosis DNA strands are separated into templates (parental DNA) new strand compliments template Summary of Interactions Each monomer is stabilized by covalent bonds P grp has charge DNA is charged therefore, P groups should repel each other in DNA strand if too close together Solution: P strands separated a great distance (dimnimish electrostatic repulsion according to coloumbs law)

Nucleosides are not stable in water therefore, inside the DNA molecule stabilize interaction via van der waal interactions since the nucleosides are stacked closely together

Form and Function of DNA Sequence of DNA allows to Store and encode genetic information

Thermodynamics Entropy: tendency of molecules to want to maximize their motion and the room that they occupywant to maximize their freedom of motion Nonpolar molecules dont interact effectively with water (NP dont form H bonds) therefore, water forms a cage around NP molecules there is an energetic penalty for the formation of this water cage since water is tied down if two NP coalesce (come together and interact) then they shed some water molecules from water cage reduce from 14 to 9 water molecules forming the cage therefore, 5 waters are free to move therefore, gain in ENTROPY (favorable process, spontaneous reaction) key: change of entropy drives hydrophobic effect

1st law: total energy of system and surroundings is constant 2nd law: Defined with gibbs, enthalpy, entropy and temperature ==> spontanoue: - value of gibbs free energy

Applied to DNA H bonding between bases to form double helix (heat is released), van der waal interactions, bases are hydrophobic (stacked interior of helix, saved from water) these reasons make the bonding of 2 DNA strands ENERGETICALLY FAVORABLE

Acid Base Chemistry Equilibrium constant: Ka HA is monoprotic (donate 1 H) pH=pKa [A-]=[HA] in Henderson-hasselbach equation acetic acid (monoprotic): if titrate H20 with HCl, pH will decrease very quickly (adding acid to water) but in acetate solution, there is a drop in pH then a pH stabilization (buffer region protonate acetate to form acetic acid to resist pH change) then further dropping of pH when all the acetate has been consumed, then no longer in buffer region and pH drops more Buffering range: inflection point is when pKa=pH As you add more HCl, convert acetate to acetic acid

Polyprotic Acids Donate multiple H (phosphoric acid), have multiple Kas Titration: Acid+ base (NaOH) increase [NaOH], increase pH

Amino Acid Structure Functional group with pKa> buffer pH will be mostly protonated and vice versa i.e Hystidine: pKa=6 inflection point in graph is pKa

DNA structure is dependant on pH at extreme pH, alteration of DNA guanine can be deprotonated at high pH (N is deprotonated and gets charge weakens bonding of GC destabilizes DNA graph: increase pH, decrease double helixs

Protein Structure proteins are composed of 20 different monomers (instead of 4 like DNA) in DNA forms double helix irrespective of sequence of DNA (as long as strands are complimentary to one another) however, not true for proteins: i.e take AA sequence and it will fold into 3D structure but different AA sequences can not adopt identical folded 3D structures (fold differently)

Structural Representations radii of molecules represent the optimal van der waal contact distances

Lecture 3 Amino Acid and Protein Structure protein is linear polymer composed of 20 different AA monomers protein structure is hierarchical: 4 levels

Building Blocks AA: C atom (C alpha carbon) bonded to 4 different groups (amino, H, COO-, R grp) Mirror image: D and L isomers (if you try to superimpose the groups, will not match up because physical arrangement of groups does not overlap therefore, have stereochemistry) L isomer: biologically relevant isomer (naturally occur) AA have amino and carboxylic acid group: acidic pH, both groups protonated titrate to neutral pH, acid group loses H (becomes carboxylate) and NH3+ (zwitter ion, have +/- ion) basic pH, everything deprotonated

Glycine and Alanine Glycine is only AA that is non chiral since R group is H (therefore, alpha C is bonded to 2 H, therefore, not chiral) Alanine: R= CH3

Hydrophobic AA (4) Proline Cyclic AA: the side chain bonds to alpha C and loops around to form C-N bond with amine group Valine, leucine, isoleucine, methionine(has sulphur) composed of C,H,S

Aromatic AA (3) Phenylalanine, tyrosine, tryptophan (5 and 6 membered ring)

Aliphatic AA with OH grps Side chain with C and OH grp serine, threonine Polar AA (OH groups can form H bonds)

Cysteine Contains sulphur can oxidize with other cysteine to form disulphide bridge

Basic AA Have ionizable side chains Lys and Arg are + in neutral pH

AA properties continued **Asparagine and glutamine do not ionize but can accept (carboximide groups) Each protein polymer have free terminal groups (amine and carboxylic acid group) KNOW: the common pKa values of side chains (AA with Ionizable side chains slide) *histidine: side chain has pKa value = 6 this is very close to pH7 therefore, 90% of side chain is deprotonated (hasslebach equation) KNOW: full name, 3 letter abbreviations (dont need to know 1 letter abbreviation), structure (side chains), which are hydrophobic/hydrophilic, acidity, pKa

Protein Structure Bring AA together via condensation reaction (Primary structure; sequence AA) Protein folds locally into different conformations (secondary structure) helical, pleated sheet

Primary Structure DNA RNA protein : protein synthesized from 1st free amine group (1st residue) to last free carboxyl group (last residue) Form peptide bond via condensation reaction (between amine and acid) water is lost Cysteine can be oxidized to form S-S bond insulin contains 2 different chains (A and B chain) A chain includes disulfide inside chain but also sulfide bonds link 2 chains together

Partial Double bond in peptide bond (sp2 groups) forms planar state and causes trans structure Can also adopt cis state around partial double bond 2 grps will sterically clash with each other (energetically unfavorable) Exception: Proline has cyclic side chain steric clash occurs in both cis/trans state, therefore, this AA can adopt either geometry in peptide bond (whereas the other 19 AA adopt trans conformation)

Psi and Phi angles These are the angles of the rotating bonds Key: certain bonds have free rotation (phi and psi) but other bonds (due to chemical properties) are unable to freely rotate (i.e peptide bond) Plot phi and psi angles (Ramachandran diagram): find that certain regions of rotation space are energetically favorable because they prevent steric clashes of side chains (i.e dark green regions are most favored) Key: even though there is free rotation in some bonds, not all the angles are favorable

Secondary Structure Alpha Helix Single polypeptide chain adopts helical structure, all side chains face outwards (but in DNA they stack inside) 1st reside and then add 4 to 1st residue to get the 2 residues that form H bond DNA has () charged P backbone that doesnt interact with one another protein has H bonds between carbonyl and amide group (of AA that is 4 structures away) DNA: bases inside Alpha Helix: side chains face out DNA is double stranded helix protein is single stranded helix Beta Sheet Stretch polypeptide chain out align with another chain and H bond with adjacent strand

Each strand: side chains tend to face away from each other (energetically favorable, side chains face opposite directions) Pleated sheets:

Questions: parallel/antiparallel orientation of Beta sheet Lecture 4 Alpha helices AA #1 and #5 form H bond (i, i+4) Helix twists from L to R (right handed) since AA have L-configuration B-Sheets Not flat but pleated AA in chain are in opposite sides (energetically favorable to avoid steric interaction and repulsion)

B) Arrows: beginning is N terminal, end of arrow is C terminus (shows polypeptide direction); some arrows are anti-parallel or parallel Secondary Structure Loops: occur between B-strands or Alpha-helixes dont have a defined geometry (can have many different types of confirmation)

Tertiary Structure Hydrophobic AA: buried inside protein (not exposed to water) Ferritin: alpha helix structure B-barrels: made up of B-sheets inside of barrel are fluorescent proteins (that can help track tagged proteins)

Domains: single polypeptide chains folded into discrete units, domains CD4 has 4 domains (color coded in red, yellow, blue, salmon) segments of polypeptide that fold into indv units (independent of one another) neighboring folded domains can associate with each other Quaternary Structure Composed of 2 or more polypeptide chains

Protein Folding Unlike DNA, AA sequence dictates the protein folding (hydrophobic AA inside, hydrophilic outside) Take folded protein and expose to denaturants (i.e urea, guanidinium chloride) folded to unfolded intermediates dont have long half life transient intermediate, very short explains sharp difference (highly co operative) between folded and unfolded state (small different in [denaturant] causes protein to go from folded to unfolded state) As [denaturant] decreases, a lot of H bonding b/w hydrophobic AA and polypeptide chain starts to fold Disulfide bonds: Ribo A is not denatured in presence of denaturants why? Disulfide bonds create cross-links b/w different parts of the polypeptide sequence, these cross links resist denaturation disulfide bonds are covalent bonds; very rigid, stable interaction (unlike H bonds) therefore can resist denaturation How denature? Reduce disulfide bonds using B-mercaptoethanol reduce to cystein residues

Protein Misfolding and Disease Prion Diseases (PrP): alzheimers, Parkinsons aggregation (grouping) of amyloid B/alpha synuclein proteins Aggregation in neural tissue (grouping) Collection of PrP forms fibre like structure Form parallel B sheets ***

Post-translational Modifications of Proteins Modifided with various chemical groups (phophorylation etc) Occur on side chain (alter stability, function, cellular localization)

Commercial Application Artificial sweetner: aspartame dipeptide of methyl ester of phenylalanine and aspartate ingesting dipeptide (breakdown via hydrolysis) PKU: genetic defect, cant metabolize phenylalanine (avoid aspartame)

Medical Application Vasopressin: water absorption from kidney peptide drug has no amine group, argine has Disomer (why? Extends half life because protease do not recognize D-isomer, inefficient at degredation only recognize naturally occurring L-isomers)

Analyzing Protein Structure Protein Sequencing Mass Spectrometry Laser excites ion (laser hits protein, ionized), charged ion is accelerated through E field with detector at end mass:charge ratio influences how fast particle strikes detector (small particle strikes faster than larger particle) time of flight

X-ray Diffraction Proteins and nucleic acids can be crystaliized collect diffraction pattern and derive structure of protein/nucleic acid For high resolution ************** The lower the number, the more info that can be obtained (smaller the number, the more detail we can see) H atoms very small, cant be seen (unless with high resolution) therefore, infer H *

NMR Spectroscopy Get repetitive structure (not a lot of freedom of movement), see very fine detail (but only single snap shot of different conformations that molecule adopts in solution) Measure binding sites, strength of molecule to protein interaction HNMR: tells a little bit about nature of environment that H is in NOESY: spin of one H can affect spin of the other (creates peak in plot) can get distance/proximity of H and derive structure

Question: Explain resolution and X-ray structures Lecture 5 2 strains bacteria: S (capsule, kills organism) and R line if heat killed S line and R line are injected into mouse, mouse dies found that some information from heat killed S line was incorporated into R line (genetic information from S line can be transferred to R line) Watson and Crick: molecular structure of DNA Meselson-Stahl: DNA replication is semi conservative

Linked with phosphodiester linkages (RNA and DNA) between 3 and 5 end stable bond

At 2` C: DNA has H, RNA has OH Nucleotide: sugar, base, Phosphate Ribose (2`C OH) and deoxyribose (2` C H) RNA is more unstable than DNA OH- ions attach OH to remove it to more O- will attach P to dissocate phosphodiester linkage (it can be a target of alkaline hydrolysis) ** Add P to nucleoside nucleotide (sugar+base+P)

Nitrogenous Bases Purine (adenine, guanine); Pyrimidine (cytosil, thyrimine, uracil) MEMORIZE STRUCTURES Max absorbance occurs at 260nm

DNA structure (Watson and Crick) A:T, G:C ratio close to 1 A+G (purine) =T+C (pyrimidine) X ray diffraction: Dna has helical structure One turn of DNA helix is approx 34A Diameter of DNA is constant (~20A) always purine+pyrimidine GC: 3 H bonds (tighter) than AT:2 H bonds

Major/Minor Grooves The other side of major groove is minor groove (cross section) Major groove has wider section of circular DNA then minor groove since major groove is wider than many DNA binding proteins bind to major groove

Semi Conservative 15N is heavier than 14N separate according to density Original molecule: 100% 15N put into solution with 14N to synthesize new DNA

DNA double helix can be melted Melting: strand DNA separation break H bonds monitor via Hyperchromic effect (detect observance of UV) Separate strands, bases are exposed DNA able to absorb MORE UV (use UV as measure of DNA) absorbance increases as DNA melts (260 nm)

Melting temperature is where 50:50 single:double strand Tm= only double helical structure remains Water stabilizes DNA via forming H bonds

Single stranded DNA can form stem loop artificial

SS RNA stem is naturally occurring (critical for biological function)

DNA replication: semi conservative; one strand DNA used as template to synthesize new DNA DNA polymerase I adds nucleotides Needed: all 4 DNPs, Mg (activation DNA poly I), template DNA strand, primer DNA strand (short DNA fragment complimentary to template strand required to start polymerization) 5 to 3 2000 nt/second (DNA poly I) very accurate (have nuclease to proofread) can recognize, remove and replace wrong nt

Lecture 6 Last week: look at DNA structure and replication DNA carries genetic information (in both vertebrates and non vertebrates) (sometimes RNA is used to carry genetic information) Exception: RNA virus replicate RNA using replicase protein Exception:Retrovirus: reverse transcriptase generates DNA from RNA (complimentary to each other, therefore, form RNA/DNA hybrid) then digest RNA to leave SS DNA replication to get DS DNA integrated into host cell genome to replicate viral DNA

RNA (3 types) rRNA: most abundant, component of ribosomes where proteins are synthesized, largest RNA in body sediment coefficient: speed of sedimentation (bigger S means larger molecule) tRNA, rRNA are directly involved in protein synthesis mRNA provides genetic information, heterogenous mass

RNA Synthesis RNA polymerase synthesizes RNA ; Mg is necessary (similar to DNA) used to anchor RNA and incoming nucleotide (Mg critical for enzyme to function) Difference from DNA polymerase (can remove wrong nucleotide, proofreading) is that RNA poly does not have repair function error prone

RNA transcription Differences from DNA synthesis: template used is ONLY the antisense strand (non coding strand) of DNA (3` to 5` orientation), primer is not required (but DNA needs primer to start synthesis) therefore can start synthesis with only nucleotides, uracil replaces thymine Phosphodiester bond : hydroxyl attacks triphosphate group (2 P leave and H20) Sequence of RNA is complimentary to DNA sense strand/coding stand (except U replaces T) Bases are complimentary to each other

Promoter Sequence

Where does RNA synthesis start on DNA?

Prokaryotes In E.Coli: upstream of start site (of RNA synthesis) there is -35 region and pibnow box (-10) on DNA template Promoter site Sigma subunit of RNA polymerase finds two regions (-10,-35) in promoter sequence, stops, allows RNA synthesis

Eukaryotes CAAT and TATA box are upstream of start site of RNA synthesis Difference from proks: these sequences are not directly recognized by RNA polymerase, instead they are recognized by transcription factors (TATA box binding protein, TBP) then RNA polyermase is recruited to initaiton transcription from start site TF recognize promoter sequence, recruit polymerase, transcription initiated Enhancer: does not initiate transcription, but enhance/suppress transcription There are many molecules involved that control RNA transcription in Euks mRNA is modified in euks: 5`cap (cap structure recognized by ribosomes for protein translation) , 3` poly A tail (may increase mRNA stability), splicing (remove introns, non coding regions, which interrupt coding sequences, exons) mature RNA is generated, protein is now synthesized

Translation -

mRNA provide genetic information, tRNA reads the info presented in mRNA (use anticodon to bring correct AA to be incorporated into protein), rRNA composes ribosome (2 subunits) where protein is synthesized

Proks protein translation starts from AUG sequence after purine rich sequence (recognized by ribosomes where initiation complex is formed; purine rich sequence base pairs with rRNA to initiate translation) AUG is first codon (formly- methionine is first AA)

Euks ribosome recognize cap structure, AUG (methionine, not formulated)

key: in either case, ribosome recognizes different structures (purine rich or cap) Genetic Code how many nucleotides in codon?3nt=1 codon= AA non overlapping, no punctuations (no gaps, continuous), degenerate (3rd nt in codon can be different since there are 64 different possible combination and only 20 AA) different codons can generate same AA helps resist mutations ***methionine and tryptophan are only encoded by only one codon 3 stop codons: dont encode any AA

Summary: Nt is unit, RNA and DNA are polymers of nt 2` position: OH in RNA, no OH in DNA RNA is relatively unstable compared to DNA due to OH A:T and G:C is close to 1 in DNA, Watson-Crick: antiparallel, major groove/minor groove, stabilizing effects helical structure lost= Tm melting of DNA monitored by hyperchromic effect Meselson-Stahl: semi conservative (need DNA polyermase, Mg, template DNA, primer, nt) 5` to 3`, both strands used for DNA replication (which is why it is semi conservative) DNA/RNA can form stem loop structure DNA (and sometimes RNA, RNA virus or retrovirus) can carry genetic info RNA only uses anti sense strand as DNA template Sigma subunit (proks) vs. TF (euks)

Example of Exam questions 1. Uracil is present in d) 2. T or F? False (DNA poly cant use the top strand as primer because the direction is wrong DNA does not replicate from the 3` to 5`) Chapter 5 We can now generate insulin using biotechnology Purify insulin from animals (dont need to kill animals or purify animal stuff) OR take human DNA to generate human insulin protein directly

1. Restriction Enzymes Restriction enzymes digest foreign DNA in a sequence specific mannerenzymes are found in bacteria (proks) Viruses infect bacteria to kill bacteria, therefore, bacteria have system to fight infection (primitive immune system) bacteria therefore have enzymes to cut viral DNA Methylation protects** Enzymes recognize palindrome sequences to digest, cleaves via hydrolysis of phosphodiester bonds Plasmid: digest DNA, fragments put on gel (smaller fragments move faster and travel further) Can help you see if there are mutations present

2. Blotting techniques Identify genes of interest via hybridization Run digested DNA fragments (via restriction enzymes) denature to SS DNA (via blotting) so can hybridize to added DNA probe helps identify if your gene of interest is present in DNA

3. DNA sequencing Directly determine nt sequence in DNA fragment Use dideoxynucleotides (lack 3`OH) for phosphdiester bond, need 3`OH group to attack P but if use analog, cant attack P, therefore, DNA synthesis stops (termination)

In addition to normal nt ALSO include dideoxy analog (i.e dATP) can generate various lengths of DNA ending with A and determine where A is located Place sequencing results on gel electrophoresis : read from 5` to 3` end (shorter ones are read first) i,e TCCTAAG Now we know the exact sequence of DNA

Lecture 7 4. Molecular Cloning Amplify gene of interest using a vector (vehicle) like a plasmid (circular DNA with origin of replication, capable of replicating in a cell by itself and host cell replication system) Cut plasmid with restriction enzymes, ligate gene of interest (vector and ligate need equivalent restriction enzyme sites, same over hanging sites need cohesive sites, ends are complimentary to each other) then amplify plasmid by putting into bacteria, antibiotic resistance gene (if plasmid is in bacteria, then bacteria can grow on this medium i.e penicillin)

5. Solid phase ******* solid phase synthesis of a DNA chain by the phosphate triester method 6. PCR Exponentially amplify target gene only (not plasmid too) Heat strand to separate DNA strand anneal primer to 3` end of SS DNA (so synthesis DNA goes from 5` to 3` direction) synthesize new DNA with thermostable DNA polymerase (since cycle uses heating step, isolated from bacteria growing in hot spring/boiling water resistant to high temperatures and still stable dont need to add DNA every time, shortens time and increases efficiency) Need to know the sequence of the flanking sequence of target DNA to make primers Since annealing of primers is based on H bonds, then you can control the specificity by changing the annealing temperature (i.e at high temp, primer A only binds to specific sequence but lower temp might allow primer mismatch) change temp, alter specificity Adding substances that alter H bonds can control specificity of PCR (i.e salt concentrations) After 1st cycle: have 4 strands 2nd cycle: heat to separate, cool, excess primers still present, the new green strand will just contain the target sequence (using templates of new strands) 3rd cycle: as you increase the cycles, increase the short strands therefore, amplify target gene fragments Used in medicine : identify viral presence Used in forensics: identify individuals HLA or STR sequences are very specific to each person (very diverse in population) having identical HLA is very little between two people Most groups are protected (I.e 5` ATP and amino group are protected so they do not react) Only group that is active is the 3` thing in red circle (NH2) reacts with OH 3` to form phosphodi Once bond is formed, the

7. RNA interference

Specifically destroy mRNA by introducing dsRNA containing target gene sequence dicer (ribonuclease) cleaves into siRNA (short, interfering RNA) SS siRNA interacts with target RNA via annealing to form dsRNA RISC formed and target RNA is cleaved, destroyed Used in cancer therapy to target oncogenes

8. Transgenic Transgenic mice contain extra genetic info (overexpress certain genes) Helps us understand how these introduced foreign genes function in living animals How to make transgenic mice? Introduce promoter in gene, inject into egg, Key: understand role of gene in living animal (can introduce ANY gene) i.e green fluorescent protein into mouse (GFP)

9.Knockout animal Opposite to transgenic mouse delete gene of interest via homologous recombination (HR) HR: Phenomena that may happen in cell (important DNA repair/diversity) DNA sequence swapping occurs in embryonic stem cells (ES cells) (young, proliferating cells) Can delete or mutate gene in genome with HR introduce construct gene (containing mutation) into cell HR MAY occur and integrate mutation into target gene via DNA swapping Mutation is now in target gene via HR inject mutated target ES cells into egg mouse now has chimeric characteristic (mix of original normal ES cells and targeted ES cells) if sperm is made from ES target cells then next generation will have all mutated gene targeted mice i.e muscle atrophy: myogenic gene deleted normal: muscle fibred present in knockout mouse: muscles are absent therefore, myogenic gene is important for muscle development leptin knockout: obese mouse, therefore, cant control metabolism of mouse

10.Microarray application of blotting techniques and kind of screening method that examines the expression levels of all human genes (which genes are up or down regulated) use gene chip to compare the expression of genes b/w normal and tumor genes prepare probes from tumor and normal (color green or red) then combine equal amt DNA and hybridize to chip if equal lvl expression, equal amt red and green hybridize and show yellow if gene over expressed, then more red hybridizes to chip so show red color (tumor) key: intensity of spot on chip is indicative of relative expression

11.iPS cells indiced pluripotent stem cells terminal differentiated means cells can no longer become anything else BUT use this technique, can covert to cell that can become anything (stem cell) only 3 genes necessary to convert adult fiberblast to iPS which can become anything (i.e nerve, liver or myocardial cell) Prepare fibreblast (adult skin cells) convert to iPS to cardiac cell cure heart failure? Regenerative medicine

Summary 1.restriction enzymes recognize palindormic sequences 2.southern: DNA probe detects DNA, Northern: detect RNA using DNA probe 3.DNA sequencing: key is to use dideoxynucleotide analog of nt which (lacks 3`OH and can terminate DNA synthesis) to generate DNA fragment containing nt 4. cloning: using plasmids to amplify gene of interest 5. Solid phase: method to synthesize oligiont DNA without DNA polymerase 6.PCR: thermostable poly used to amplify target DNA (medical, forensics) 7. target mRNA using dsRNA 8. transgenic mice (overexpress), knockout (dont hace it) analyze function of gene in vivo (in living animal) 9. microarray: screens gene expression based on hybridization 10.iPS mature cells can be converted to become puripotent Sample Exam Question: Plasmids used in recom DNA typically: D Lecture 8 Hemoglobin and myoglobin are not enzymes but bind and transport oxygen in blood Mb: simplest oxygen binding protein, protein consists of alpha helixes ONLY, chemical group (heme group consists of ring system with 4 N in center coordinate around Fe oxygen binding sit is the heme group and oxygen directly coordinates with Fe) Hb: oxygen cant diffuse through skin fast enough, therefore, if no circulatory system then out internal tissue would die from oxygen deprivation Oxygen has low solubility there has to be a way that RBC can transport large quantity of oxygen therefore, use Hb which has larger solubility than O2 50 fold greater than oxygen solubility therefore, body can concentrate O2 in blood by binding to Hb Hb: transport O2 in blood stream from lung to tissue Mb: provides oxygen reservoir (when exercising, need oxygen Mb can release oxygen due to demand) Hb: bind O2 with high affinity/tightly so it can transport it from the lungs BUT low affinity in tissues so it can unload O2 how do this? two different Hb states with diff binding affinities that can be altered Fe in heme ring system can exist in oxidized state (Fe3+) which can be reduced by e- to oxymyoglobin (Fe2+) Fe2+ binds to O2 oxy__globin formed, can form resonance structure known as superoxide anion with Fe3+ superoxide is problematic, highly reactive and can cause oxidation of proteins/DNA/RNA which causes damage How prevent superoxide formation is Mb/Hb? H bond with distal histidine prevents formation/release of superoxide allows Hb to transport O2 without formation of superoxide

Proximal histidine: in deoxyHb (O2 not bound) the Fe doesnt sit in the plane (slightly below plain) O2 binds to Fe, Fe moves into plane of ring system that is heme group When O2 binds, Fe moves into ring and maintains coordination with proximal histidine group (helps stabilize binding of Heme grp) Refresher: AA sequence, local conformation of backbone, folding of alpha helices in one subunit of hemoglobin, all 4 subunts assemble #3D to form hemoglobin 2 alpha, 2 beta subunits Mb is single subunit with single heme grp, Hb are 4 subunits with each one bound to heme grp

Oxygen binding to Mb Hb: signoidal function (S shape) P = 25 torr Mb: P = 2 torr Different binding affinities, Mb binds very tightly in lungs but then doesnt release it Cooperativity: change in behavior and binding affinity and conformational change (in each subunit) which stimulates heme to release oxygen in tissues (these two states causes the change from high to low binding affinity) : Hb SO what? When Mb bound which O2 travels from lungs to tissues, it loses 7% of oxygen (change in oxygen saturation) Hb: changes fractional saturation from lungs to tissues by 66% (more oxygen released) No cooperativity in Hb: bind O2 weakly (therefore pick up small amount but release very easy) Deoxy state: T state subunits arranged in such a way that they are not contacting each other, therefore, there is a pocket in the middle O2 binds, conformational change, subunits relax from tense state and pocket closes in relaxed state to oxy state (S) O2 binds to Fe, Fe shifts into plane of heme grp, proximal histidine coordinated to Fe also shifts, shifting of histidine side chain causes conformational change in helix, helix interacts with neighboring subunit at interface, stimulates conformational change to R state (higher affinity binding state) of Hb

Concerted model All or nothing model: exists in either T or R state R state favored as O2 binds but T state favored when no O2

Sequential Model Start with deoxyHb, O2 binds, subunits change in conformation, signifies increase in binding affinity, each binding of O2 stimulates conformational change until saturated

**Hb doesnt strictly follow either one or the other model

2,3-BPG very high negative charge (2 P grp and 1 Coo) stabilizes in T state 2,3-BPG binds to pocket in T state stability Hb with no 2.3-BPG: binding curve similar to Mb (not a lot of cooperativity), fractional change 8% Hb with 2.3-BPG: signoidal curve, S shaped , 66%

In absence, mostly in R state 2.3-BPG helps balance between T (responsible for releasing oxygen)/R state

Fetal Hb Different subunit composition, 2 alpha and 2 gamma Gamma vs. beta (adult) gamma: binds O2 more tightly go from single cell to baby in 9 months, need high [O2] to fetus therefore, at placental interface the oxygen goes in one direction from mother to fetus

Exercise: pH drops, increase [H+]/CO2, shift R, favor T , decrease affinity (release more oxygen) protonation of hisitidine residue, forms H bond with Asp, electrostatic interaction favors T state Free amine grps at beginning of polypeptide react with CO2 to form carbamate

Formation of carbonic acid and H+ makes interior of RBC acidic favros change from R to T state and causes release of O2 from RBC

Sickle Cell anemia Mutation in Beta (single cell mutation) Mutation from Gl to Val causes hydrophobic interaction, Hb aggregate causes RBC to become elongated, sickled and can clog or plug arteries (stroke etc) Heterozygous for mutation causes resistance to malaria

Lecture 9 protein based enzymes dont need to memorize enzyme classes active form of enzyme that requires cofactor, holoenzyme coenzymes, metals : cofactors (there are two broad classes) in presence of enzyme, reactions are incredibly efficient (shorter half lives) capable of accelerating reactions Free Energy (G): depends on only reactants/substrates and products, doesnt provide info about rate of reaction ONLY spontaneity Even though delta G not is +, depending on the [] of substrate and product, the rxn can be favorable (G<0) [] reactant/product AND delta G not are both indicative if rxn will proceed favorable With enzymes, the product/substrate reach equilibrium (level off) Favorable reaction but not spontanoues since has TS enzymes reduce energy barrier for rxn (bind to TS and help stabilize energy of TS) enzymes do not change the energy of product/reactants (stay constant) but do alter energy of TS

Active site is usually confined to cleft or pocket on enzyme Lysozyme: cleave cell wall of bacteria (then dies) 3D structure: well defined cleft where cell wall binds and is hydrolyzed in primary sequence, the active site residues are scattered throughout . Adopt 3D structure, residues are brought close together in active site Lock and Key: shape of substrate and active site fit together Induced Fit: binding of substrate causes active site change, promotes association

Chymotrypsin Protease: enzymes that hydrolyze other proteins by activate H20 to cleave peptide bond, polarize carbonyl grp Recognizes the large hydrophobic side chains, immediately cleaves the peptide bond following the AA forms amino and carboxyl component Active site of chymotrypsin: catalytic triad (asp, his and ser residue) H+ from Ser hydroxyl grp can be transferred to His (produces reactive alkoxide ion which is responsible for catalyzing cleavage of peptide bond) **memorize residue numbers** 8 steps to rxn Alkoxide ion forms, nucleophilic attack which forms tetrahedral intermediate negative charge on Oxygen binds to oxyanion hole (pocket in enzyme active site) Gly 193 and Ser 195 have amide grps that form H bonds that stabilize negative charge on Oxygen in tetrahedral intermediate

Substrate binds to active site of enzyme, bulky hydrophobic AA side chain binds in pocket , oxyanion hole is where H bond forms between carbonyl grp Alkoxide ion forms, negative charge on Oxygen is stabilized in oxyanion hole (H bonds from amide of gly and ser) Peptide bond breaks and portion, acyl enzyme intermediate (serine hydroxyl grp is acelyted) Water molecule enters active site and forms H bond with deprotonated side chain of amitosol and histidine lone pair e- of oxygen from H20 attacks C in carbonyl grp new tetrahedral intermediate formed (negative charge on O stabilized by gly and ser H bond in oxyanion hole) Rearrangement of bonds, peptide product released from active site **learn mechanism**

Specificity pocket close to active site: this pocket binds to large hydrophobic residues , binding helps orient the peptide bond that needs to be hydrolyzed Overall structure of pocket are similar but substrate Elastase: valine residues plug up pocket and only allow small AA residues to bind Trypsin: basic

Enzyme Kinetics Enzyme+substrate associates to form ES complex (can dissociate or react to form enzyme and product) Initial rate of reaction, Vo (V knot)

Vmax: as more substrate molecules are added to rxn, enzyme molecules become saturated with substrate all enzymes now exist in ES form and constantly convert substrate into product Km ( Vmax): gives an idea of how tightly enzyme binds to substrate, if Km is low then tight binding , high binding affinity (doesnt take a lot of substrate to achieve Vmax) Kcat K2 is rate limiting step , K2=Kcat Catalytic efficiency: Phe has largest efficiency

Allosteric: The binding of one substrate, can effect the binding of another