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Biochem Exam 3 Study Guide

by: Kiara Lynch

Biochem Exam 3 Study Guide BIO 412-01

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Kiara Lynch
La Salle

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These notes cover the information on exam 3. These notes include proteases, carbonic anhydrases, restriction endonucleases, myosin(ch 9), regulatory strategies (ch 10), lipids and cell membranes (...
Stefan Samulewicz
Study Guide
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This 29 page Study Guide was uploaded by Kiara Lynch on Tuesday February 2, 2016. The Study Guide belongs to BIO 412-01 at La Salle University taught by Stefan Samulewicz in Summer 2015. Since its upload, it has received 97 views. For similar materials see Biochemistry in Biology at La Salle University.

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Date Created: 02/02/16
BIOCHEMISTRY EXAM 3 STUDY GUIDE CHAPTER 9 Proteases  HIV protease and indinavir complex (inhibitor) o Protease inhibitors are used as drugs and are specific for one enzyme  Don’t inhibit other proteins within the bodyprevents side effects Carbonic Anhydrases- 7 types  Increase rate of reaction  Dehydrate HCO3- in blood to form CO2  Convert CO2 to HCO3-  accelerate CO2 hydration  CO2 + H2O carbonic acid  bicarbonate ion + H+ o Event without a catalyst, the reaction proceeds moderately fast. o Reverse rxn is faster  Zn atom helps create a nucleophile out of water o Essential for catalytic activity o Bound to 3 His residues and water o In cleft near center of enzyme  Effect of pH on carbonic anhydrase activity o Reactions/second vs pH o Histidine pH around neutral o pH 8- enzyme maximally active at high pH o deprotonated form of group participates more in catalysis  Carbonic anhydrase with Zn o Histidine holds Zn atom in place o Deprotonated H2O becomes a strong nucleophile; H+ byproduct in center  Zn can create strong nucleophiles o 1 million x per second – 1 million H+ in active site per second o Binding of H2O to Zn lowers pKa of water from 15 to 7  Water loses proton at pH 7  [OH-] increases o Adjacent hydrophobic patch- binding site for CO2 o Zn  H2O to OH  CO2 to active site  react with OH  HCO3-  release  bind H20  Analog- synthetic molecule, looks like 4 histidines o Capable of binding Zn  Zn bound to ligand and 1 water molecule o Zn can create a nucleophile from water o Accelerated production of carbonic acid from CO2 and water o Accelerates hydration of CO2 100 fold  Proton Shuttle o Rapid regeneration of active form of enzyme o Water deprotonated  protonation of Zn bound H2O o K needs to be 1 o Protons in active site  not enough room (1 mill/sec) o In absence of buffer, equilibrium constant is highly uneven  doesn’t work physiologically o B-buffer effect on deprotonation  K becomes 1  Highest rates of CO2 hydration require presence of buffer  buffer components participate in reaction  Increased concentration of buffer  increased CO2 hydration  More places for proton to go  reaction rate increases o Histidine Proton Shuttle  Active site in center of molecule- no buffer there  Gets protons from center out to enzyme surroundings  Shuttles to other histidines and that histidine releases H+ so protons aren’t in the way  His takes H+ from Zn bound water molecule and makes it a nucleophile  buffer removes H from histidine  Convergent evolution- Zn atoms o Type of carbonic anhydrase- trimer  Beta helices and 3 fold symmetry o Zn bound water molecule bound to 3 histidines Restriction Endonucleases (enzymes)  Evolved to fight off viruses  defense mechanisms produced by bacteria  EcoRV- ecoli restriction enzyme #5 o Palindrome- same sequence but in reverse order; antiparallel  Allows for copying and passing on info o Substrate- double stranded DNA  Need to cut twice in same spot to break it  Need same substrates; look for pallindrums o Enzymes there all the time  Cut DNA – no nucleus in bacteria  Difference between viral and bacterial DNA  Prevents enzyme from cutting host DNA o Methyl group- methylates own DNA o Changes shape of substrate so it doesn’t fit into active site o Hydrolysis of phosphodiester bonds  Ribo- sugar  Link together sugars 5C–5’ 3’–3C links on other end  All restriction enzymes catalyze hydrolysis of DNA phosphodiester bonds o Phosphoryl group at 5’ end  7 lbs of bacteria in body  bacteria produce restriction enzymes  cut viral DNA  prevent integration  defense mechanism  6 mer EcoRV cleaves  Create new 3’ and 5’ end  1 step vs. 2 step hydrolysis o 2 step hydrolysis  Covalent intermediate; Releases part of product  Hydrolysis of intermediate  other part of product and molecule with OH to break bonds o 1 step hydrolysis  Direct hydrolysis- creates bond that holds backbone together o In-line displacement reaction o Intermediate has trigonal bipyramidal geometry- rearranged so no steric hindrance o Product- invert stereochemical conformation, RS o Blunt end cut  Cuts phosphodiester bond in the middle of the pallendrum  Restriction enzymes don’t always do that  usually cut at both ends o Need to look at arrangement of groups on phosphate  Replace one oxygen with sulfur so you can tell the oxygens apart  Labeling with phosphorothioates  Nonbridging oxygen is replaced with a sulfur atom o Stereochemistry of cleaved DNA  Cleavage of DNA by EcoRV endonuclease causes inversion at the phosphate atom  Leads to hydrolysis by water’s direct attack- 1 step reaction  You can also you heave oxygen (water) to tell the oxygens apart 18  Ex: 2 normal oxygens, 1 sulfur, and 1 O  Creating a nucleophile from water o EDTA  Metal chelating- binds tightly to metals  Don’t want nuclease to cut DNA  Mg ion binding site in EcoRV endonuclease  Helps activate water and position it to attack the phosphate atom  Scissle bond- bond that is broken  Asp74 attracts a proton from water  Mg creates nucleophile  Structure of recognition site of EcoRV endonuclease o Catalytic apparatus in cognate DNA  specificity o Imaginary axis- rotational symmetry  Binding sites on both sides (dimer) o Inverted repeat  2 fold rotational symmetry  Subunits of restriction enzyme o Kink  If DNA sequence is a pallendrum, it forms kinks at the Mg binding sites  Makes it easier to break bonds  Hydrogen bonds between amino acid side chains  Cognate DNA o Increases binding energy o Drives DNA distortions to form catalytic complex o Greater binding energy of EcoRV bound to cognate DNA vs. noncognate DNA o Enzyme + nonspecific DNA  less enzyme DNA interactions, nonspecific complex o Enzyme + cognate DNA  more enzyme DNA interactions, DNA distortion, cognate complex (catalytically competent)  Methylation of Adenine o Blocks formation of H bonds between EcoRV and cognate DNA and prevents their hydrolysis o Prevent own DNA from being cut  Make substrate so that it doesn’t fit perfectly into active site  Add methyl groups- take up space  Prevents formation of H bonds (not a good substrate)  4 Conserved structural elements o Make active site that allow protein to cut o EcoRV, RI, and BamHI all have the same 4 elements but cut different substrates Myosin  Harness changes in enzyme conformation to couple ATP hydrolysis to mechanical work  Mechanoenzymes  ATP +H2O  inorganic phosphate P + ADP o Hydrolyze gamma phosphate  Elongated structure of muscle myosin o Dimeric- 2 globular ATPase domains per dimer  Myosin ATP complex o ATPase domains  Change in location of globular proteins relative to fiber  Metal (Mg) bound to ATP- binds to phosphate and orients them so it can fit into the active site o Formation of transition state for ATP hydrolysis is associated with substantial conformational change o Myosin-ATPase transition state analog  Treat myosin ATPase domain with ADP and Vanadate in the presence of Mg  Vandium ion slows the reaction so it can be seen taking place  Vanadate- coordination to 5 oxygens, 1 from ADP  2 residues that bind Mg- Serine  Instead of metal helping create a nucleophile, Serine is used  Facilitating H2O attack  Water attacks the gamma phosphoryl group of ATP  Deprotonated by hydroxyl group of Ser 236  Deprotonated by oxygen atom of gamma phosphoryl group  H2PO4- product  Water in  not nucleophilic o Serine  not a good nucleophile either o ATP acts as its own base  Takes proton from serine  Serine takes proton from water  OH attacks phosphate  Myosin conformational changes o Large conformational change at carboxyl terminus of domain (25 angstroms) o Small (2 angstrom) conformational change to active site  Causes big change at surface between the fiber and globular regions o Energy is transferred into mechanical work o Altered conformation of myosin persists for a substantial period of time  O18 substituted to track incorporation of solvent oxygen into reaction products  1 oxygen from water and 3 initially present in terminal phosphate group of ATP  Reversible hydrolysis of ATP within myosin active site o Hydrolysis of ATP o ADP + Pi o Phosphate rotation within active site o Reformation of ATP containing O2 from H2O  Myosin family has P-loop o Loop that reacts if the 3 phosphate is present o P-loop NTPase domains- conserved domains with inner surfaces of ribbons and P-loops o Adenylate kinase, transducing (has alpha subunit), ATP synthase (has beta subunit) CHAPTER 9 SUMMARY  Carbonic Anhydrases make a faster reaction o Catalyze reaction of water and carbon dioxide to form carbonic acid  Up to 1 million times per second o Zn ion is crucial at active sites  Binds water and promotes deprotonation  OH forms at neutral pH  Attacks CO2 to form bicarbonate ion HCO3-  Proton shuttle to transfer protons to a buffer  Restriction Enzymes catalyze highly specific DNA-cleavage reactions o Substrate specificity o Substrate distorted to generate Mg ion binding site between enzyme and DNA o Mg binds and activates water to attack the phosphodiester backbone o Discrimination  Binding with different affinities  Bind many substrates but promote chemical reactions efficiently for only 1 o Restriction enzymes are prevented from acting on host DNA by methylation of key sites (blockage)  Myosins harness changes in enzyme conformation o Couple ATP hydrolysis to mechanical work  Catalyze hydrolysis of ATP to ADP and Pi  ADP and vanadate (VO ) 4imic transition state for ATP hydrolysis bound to myosin ATPase domain  Dramatic conformational changes  drive molecular motors o P-loop NTPase enzyme CHAPTER 10: Regulatory Strategies Regulatory Strategies  Allosteric control o Enzyme changes shape o Enzyme on  R state o Enzyme off  T state  Isozymes o Copies of genes in genome that produce basically the same enzyme o Slight differences in genes  slight differences in enzymes o Put control at gene level o Change individual amino acid in coding region slightly different Km (binds substrate differently) o Promoter proteins are responsible for transcription  Reversible covalent modification o Kinase- adds phosphate (usually from ATP)  turns on  ATP- phosphoryl donor  PKA- ubiquitous eukaryotic enzyme that regulates diverse target proteins o Phosphatase- removes phosphate  turns off o Conformational change o Catalytic properties altered by covalent attachment of modifying group  Proteolytic activation o Produce protein in inactive form (proenzyme) o Expose to protease  becomes functional  turns on  Cuts 1 or 2 peptide bonds (hydrolysis)  conformational change  Irreversible binding of specific inhibitory proteins  Can’t turn back off (can’t reform bonds) o Zymogens or proenzymes bond inactive precursors o Generates digestive enzymes (chymotrypsin, trypsin, pepsin) Allosteric control  ATCase o Distinct regulatory sites- binding of small molecule o Cooperativity o Involved in 1 step in multistep process of production of CTP o Condensation of aspartic acid and carbamoyl phosphate to form N- carbamoyl aspartate  2 substrates combined to form 1 product  Once combined into product, CTP forms  Committed step  Want to regulate this protein so you can turn on and off the whole pathway  Feedback inhibition- product of pathway goes back and turns pathway off o Multisubunit enzyme, multiple polypeptides  CTP inhibits ATCase- allosteric inhibitor  As [CTP] increases, rate of N-carbomoylaspartate formation decreases  As [Asp] increases, rate of N-caromoylaspartate formation increases  Sigmoidal curve, cooperative binding possibility  Allosteric o Modification of cysteine residues  Ultracentrifugation studies of ATCase  Fill ultracentrifuge tube with viscous material (sucrose or cesium chloride)  Separate molecules  Purified CTPase- treat to break disulfide bonds  Layer on top of dense material, spin  Settles at particular place based on mass  Protein concentration vs distance migrated  Native conformation c6r6, 1 peak  Treated enzyme, 2 peaks- r2 and c3  K pairs bound ATP but had no enzymatic activity  C triplets have binding sites for substrates and had enzymatic activity but CTP did not bind  Interaction of subunits in native enzyme produces its regulatory and catalytic properties o Structure of ATCase  Quaternary structure  2 catalytic trimers  Each chain interacts with a c-chain through Zn domain o PALA  Nucleophilic attack by amino group of Asp on carbonyl carbon of carbamoyl phosphate  intermediate  PALA  Bisubstrate analog  Competitive inhibitor of ATC  Reaction happens too quickly and can’t freeze it halfway  Intermediate substrate analog- molecule that looks like intermediate but is missing important components for the reaction  Gets into active site  T to R state  Prime with PALA- freeze in R state  x-ray crystallography o Active site of ATCase  Residues from 1 c chain and adjacent c chain contribute  AA side chains involved in holding two substrates o T to R state transition  T state- inactive- compact  R state- active- expanded  2 trimers connected by 3 dimers  Trimers pull apart by 6 A and rotate around axis 10°  CTP binds to specific sites on R dimers  wedge and hold open active site  R  Weak interactions  reversible  2 lobes on hinges  240 loop o Too far apart to form H bonds; loop causes active site to -become smaller so reaction can occur  Bonds between lower and upper trimer  T  Bonds (hydrogen and electorstatic) broken, hinge closes, lobes come together, internal bonds R  Rate of N-carbamoylaspartate formation vs. [Asp]  High Km- tense  Low Km- relaxed  As [substrate] increases, equilibrium shifts from T to R; rise in activity  T- activity low  R- wasting energy; making CTP all the time  T + R- sigmoidal o Concerted  All subunits change at once- all 12 subunits move/stretch as 1 Isozymes  Allows for more complexity evolutionarily ___________ gene that codes for enyme ___-___-__ __-___-___-__ mutations get replicated in genome (progeny)  Same reaction mechanism (substrate and product)  Mutations cause different characteristics (Km- ability to bind substrate)  Promotor region  copied  mutations o Alter when and where gene gets turned on and off o 1 version becomes better for 1 tissue than another o Catalyze same reaction in different conditions or times  T state is favored by CTP binding  R state is favored by substrate binding  Effect of ATP on ATCase kinetics o ATP- allosteric activator of ATCase o R- easier for substrate to bind o Curve is shifted to the left; formation rate of N-carbamoylaspartate is faster as [asp] increase o Concerted model  Add ATP more active enzyme  Imbalance- not making enough CTP  Competes with CTP for binding site on R subunit  Keeps enzyme relaxed  Ex: lactate dehydrogenase (when in O2 debt) o LDH catalyzes reaction o Pyruvate  lactic acid o NADH deprotonated  NAD+ o 2 versions- every cell has a gene for both but not everyone produces both  Heart of rat o Heart (H) 4 subs  Higher substrate affinity  Works better under aerobic conditions o Muscle (M)  In skeletal muscle  Opposite characteristics  Low substrate affinity  Lets lactic acid build up  can poison cells (causes pain so you stop  defense)  Works better in anaerobic conditions (low O2 levels) o Gives up on legs before heart o Types of LDH present in rat heart from before birth to adulthood  M- while in mother; takes O2 from her; on edge O2 wise  H- out in the real world where there is more O2 o LDH content varies by tissue  Heart is mostly H4  Tissues that need more O2  H  Dictates which isozyme is produced Covalent Modification  Cascade- trigger something with little enzymatic activity  activate 10 each big response  Reversible phosphorylation o Phosphate of ATP  Ser, Thr, or Tyr residue (all have OH)  Replace H with phosphate and 3 oxygens  takes up space  2 negative charges  Leads to allosteric changes o Protein kinases add phosphates turn proteins on o Prtotein phosphatase removes phosphate and replaces with hydrogen  Phosphate is recycled to ADP to make ATP o Kinases react with ser, thr, and tyr side chains o When phosphorylated, overall system energy goes down but proteins free energy goes up which allows the enzyme to become active  Free energy is high when phosphate is still on ATP o Reversible phosphorylation intracellular signaling  Activate proteins still inside cell  Can be deactivated  cAMP- cyclic adenylyl cyclase o startled  adrenal gland releases adrenaline (signaling molecule)  binds to receptors on muscles  fight or flight response  Signal transduction video- o Extracellular signal to intracellular signal o Used when molecule can’t pass directly through membrane o Epinephrine molecule binds to receptor protein (communication between outside and inside) change in shape  receptor activated and has binding site; binds with G prtoeins (ability to bind GDP and GTP; GDP inactive, GTP active)  activates G protein  G protein binds to receptor protein and exchanges a molecule of GTP for GDP  release of G protein in active form  binds and activates adenylyl cyclase  converts ATP molecules to cAMP which acts as secondary messenger to send signal to inside of cell  cAMP binds to protein kinase A (PKA) which further amplifies the message liver cell converts glycogen into glucose  Goal: stored energy (sugar) chop it up  glucose  broken down for energy  Happens instantaneously; simple diffusion o Cascade- useful for a fast response; original signal is amplified; low energy used; 1 molecule of epinephrine can stimulate big response o Digestion- inactive form of digestive enzyme – zymogen/precursor  PKA o Always there but inactive and waiting for signal o Consists of catalytic protein subunits  which most of the time are bound to regulatory subunits o Active site binds serine and threonine o Pseudosubstrate sequence- active site; almost all the right amino acids  Alanine instead of serine or threonine  Binds to enzyme but doesn’t react- inhibits o Kinase domain o cAMP (secondary messenger) causes a conformational change making the pseudosubstrate not fit anymore  catalytic and R subunits dissociate Proteolytic Activation  Sometimes you want a protein to have activity but not necessarily inside the cell o Ex: digestion- don’t want proteases in cells in active forms o Ex: blood clotting  Produced in cells  Secreted into bloodstream  Don’t want active until a blood clot is needed  There are activating proteins outside of the cell  Zymogen o Secretion from acinar cell of pancreas o Synthesized on ribosomes attached to ER  processed in golgi apparatus and packaged in granules o Signal  granules fuse with cell membrane and release contents o Proteins in membrane bound granules  need digestion  granules fuse with membrane of cell and release  become active by proteolytic cleavage  conformational change  active protein o Activation by proteolytic cleavage  Enteropeptidase initiates activation of zymogen by activating trypsin which activates more zymogens  Only a small amount of enteropeptidase is produced in active form  Activates small amount of trypsinogen to trypsin  Small amount of trypsin activates rest of trypsin  Activates all other proteases  “master protease” o Trypsin interaction with inhibitor  Free trypsin vs bound trypsin have similar structures  Don’t want protease to stay on  so a protease inhibitor is also released  binds to inhibitor and can’t be released  Lys 15 penetrates active site  Salt bridge formed with Asp 189  Chymotrypsin o Released in intestines o Trypsin binds to AA 15 and cuts after it o Pi chymotrypsin cuts itself twice; Amino acids are lost o Alpha chymotrypsin is held together by disulfide bonds o Proteolytic activation of chymotrypsinogen  3 chains of alpha chymotrypsin linked by 2 inter-chain disulfide bonds o Active and inactive forms  Overall structure is similar  Ile on surface but is hydrophobic; cuts chymotrypsin between amino acid 15 and 16 and creates a new amino acid terminus  Ile  free  goes to middle of protein  new + charge  attracted to Asp 194  Oxidation of methionine to methionine sulfoxide o Effect of smoking  Oxidation of methionine residue  Can no longer bind to elastase  Matrix depleted o Elastin  Key protein in extracellular matrix of lungs  Folds into random coil  Surrounds lungs and blood vessels (tissues that change shape)  Expands and compresses with tissue  Need to repair extracellular matrix because of constant change in shape  Elastase  Mutation- prevents binding of elastase  Elastin activity increases  Elastin becomes chewed away/depleted  Spaces expand, mucus clogs bronchioles  Old air trapped – Emphysema – hard to breathe  Blood Clotting o Clot- platelets bound in a meshwork of fibrin o System of proteins ready for cascade effect  2 pathways that lead to clot formation o Intrinsic pathway o Extrinsic pathway  Triggerd by trauma  Releases tissue factor (TF)  TF forms complex with VII  Cascade- activating thrombin o Fibrinogen  Alpha helical coiled helix with globular regions that have binding sites  Highest concentration in bloodstream  Short polypeptide made up of charged amino acids  Fibrinopeptides  charges help keep fibrin soluble and prevent polymerization  Negative charges repel each other  Thrombin- cuts off fibrinopeptides  Solubility decreases, H bonds form and polymerization occurs o Trauma/damage  thrombin cuts off fibrinopeptide  No longer soluble, won’t repel each other  Bind to globular domains  Polymerize  Cross links  covalent bonds, fibromonomers  Clots- prevent further bleeding; at the surface  Impervious to mildew and rock  Prevents infectious molecules from getting in  Fix skin under it (repair) o Domain structure of Prothrombin  Gla-Kringle-Kringle-2 cleavage sites-Ser protease  Kringle- linkers between serine proteases  Cleavage of 2 peptide bonds yields thrombin  Gamma carboxyglutamate residues in gla domain  Add extra carboxyl group allows to bind extra Ca ions  Globular- contains a lot of glutamic acid residues  Good calcium chelater (binds calcium well) o Rat poison  Contains molecules that look like vitamin K  Prevents production of carboxyglutamate  Can’t bind calcium as well  Gla domain anchors in the membrane  Brings prothrombin  No prothrombin  can’t activate fibrin at wound site  hemorrhaging o Calcium binding region of prothrombin  Calcium binds with gamma carboxyglutamate  Prothrombin in platelets  Want clot process localized  secrete antithrombin molecule o Plasmin/plasminogen and TPA (tissue-type plasminogen activator)  Enzymes responsible for scabs falling off  Plasmin is activated by TPA  Snips through alpha helical fibers holding clot to platelets  When a clot is formed, fibrin keeps the would shut CHAPTER 10 SUMMARY  ATC is allosterically inhibited by the end product of ist pathway o Catalyzes synthesis of N-carbamoylaspartate (1 intermediate in synthesis of pyrimidines) o Mediated by large changes in quaternary structure  CTP inhibits  ATP reverses inhibition  Cooperative binding of substrates o Substrate binds  c3 subunits of c6r6 enzyme move apart  ATCase T to R  Isozymes provide means of regulation specific to distinct tissues and developmental stages o Isozymes differ in structural characteristics but catalyze the same reactions o Fine tune metabolism to meet needs of tissue o Gene duplicates  means for subtle regulation of enzymatic function  Covalent modification regulates enzymatic activity o Phosphorylation- most common  Single kinase can act on many targets o Kinase regulating actions reversed by protein phosphatases  Catalyzes hydrolysis of phosphate groups  Many enzymes are activated by specific proteolytic cleavage o Activation of digestive enzyme and blood clotting o Zymogen- inactive precursor (proenzyme) o Zymogen activation is irreversible  Specific inhibitors of proteases exert control  Ex: antithrombin III holds blood clotting in check in clotting cascade CHAPTER 12: Lipids and Cell Membranes Features of Membranes  Sheetlike o Form boundaries, 2 molecules (60-100 angstroms) thick o Bacterial flagella would not spin without a membrane  Lipids and proteins (mediate function) o Proteins are receptors and channels and pumps  Hydrophilic/hydrophobic o Moiety o 2 parts  Hydrophilic outside and hydrophobic inside o Interact with each other  no energy is used  Noncovalent bonds o Van der Waals interactions- weak but numerous  Assymetric o Can tell outside from inside o Glycosylation- addition of sugar groups on outside of membrane  Fluid o Different molecules that are constantly moving but are held together by noncovalent bonds  Electrically polarized o Synapses o Negatively charged inside  creates membrane potential o Key role in transfer, energy conservation, and excitability o Diffuse rapidly in plane Fatty acid structure  Long hydrophobic chains with terminal carboxylic acid groups  Even numbers of C’s – usually 16 or 18 o 2 C’s enter krebb’s cycle to generate ATP  Saturation affects fluidity  Melting point is affected by length of tail and amount of unsaturation o Higher melting point shorter tails, more saturation  Unsaturation o Not optimal distance – can’t pack together tightly  Omega 3 fatty acid Components of Membranes  Amphopathic – hydrophilic and hydrophobic moieties o Phospholipids  Phosphoglyceride- 2 hydrophobic fatty acids, 3 C glycerol backbone, hydrophilic phosphorylated alcohol  Phosphatidate  Acyl groups with fatty acid hydrocarbon chains  Additional molecules added to phosphate group  Alcohols added to phosphate face out toward water  Way to make phospholid- amino alcohol with long unsaturated hydrocarbon tail (building block); add hydrophobic tail o Glycolipids  Fatty acid unit and sugar unit (glucose or galactose) that faces outside of cell o Cholesterol  Big ring structure  In eukaryotic cell membranes  Present in small quantities up to 1 cholesterol per every phospholipid  OH- hydrophilic moiety Membrane lipids  Ionized fatty acids readily form a circle but phospholipids do not  Insoluble molecules become solubilized by hydrophobic tails in circle conformation  Membranes have 2 tails o Bilayer membrane o No room to form sphere  form sheet like structures  wrap and come in contact  form compartment  Unsaturated- double bonds  fewer van der waals interactions  Outer aqueous compartment, bilayer membrane, inner aqueous compartment o Liposome, lipid vesicle, aqueous compartment surrounded by lipid bilayer  Preparation of glycine contains liposomes o Sonication  Phospholipid at bottom of beaker, glycine in water on top  Sonication- metal probe sends waves of energy in form of sound; goes through water and stirs up lipids  mix with aqueous environment  lipids associate with each other in sheets  spheres  cells  Shows how well glycine passes through membranes  Gel filtration  Glycine trapped in lipid vesicles; flush away glycine from outside; see how long it takes to diffuse out of cell and equalize concentration  Study of planar bilayer membranes o 2 aqueous compartments with divider that has a hole in it o Paint brush in lipid solution and pain across hole form lipid bilayer o Different molecules on each side of the membrane o Track movement and diffusion (can use electrode) o Permeability coefficient  Ability of molecules to cross lipid bilayer  Na, K, Cl ions slow through membrane  Glucose, tryptophane, glycerol polar but uncharged  Indole and water more hydrophobic- membranes more permeable to these molecules o SDS acrylamide gel  Membranes are 50% lipids 50% proteins  Isolation of proteins was easier  Isolate membrane and proteins come with them o Integral and peripheral membrane proteins  Peripheral- associated with membrane; don’t need to disrupt membrane to isolate; interact with polar head groups  Integral proteins- interact with hydrocarbon region of bilayer  Traverse lipid bilayer  Transmembrane o Membrane spanning helices- Alpha helix; 20 AA across membrane  Porin o Beta strands – channel protein o Beta sheets form hollow cylinders  Outside – nonpolar  Inside – hydrophilic; filled with water o Alternation of hydrophobic and hydrophilic amino acids along each strand o Periplasmic space  Prostoglandin H2 synthesis o Attached to membrane- held by alpha helices coated with hydrophobic side chains o Integral protein with hydrophobic amino acid side chains o Catalyzes formation of prostaglandin H2 from arachidonic acid in 2 steps o Damage to cells  membrane components disrupted  signaling  immune response o Hydrophobic original substrate; turn substrate into something that can circulate through blood stream to alert you are hurt; hydrophilic product o Channel through enzyme down to active site  converts from hydrophobic to hydrophilic  Aspirin o Inhibitor; covalent bond with serine 530  inactive o Hydrophobic channel that leads to active site; membrane anchoring helices o Aspirin’s effects on prostaglandin H2 synthase-1  Aspirin transfers acetyl group to a serine residue in prostaglandin h2 synthase-1  Membrane anchors o Prenyl groups- hydrophobic groups covalently attached that insert itself into membrane  Polarity scale- willingness to stay in membrane or get out o Negative numbers for transfer free energy- want to get out o Add up numbers associated with amino acid groups of 20 amino acids o Look for big number o Hydrophobic amino acid in that window of 20 o Glycophorin- protein associated with RBCs; transmembrane protein; post translational modification that makes it more hydrophilic  more soluble  Hydropathy plot o 84+  highly likely that it is a transmembrane domain o Single alpha helix in glycophorin above 84 o Porin  No strong peaks; membrane spanning beta strands (no alpha helices)  Transmembrane protein  doesn’t use alpha helices  uses beta sheets  Exception to 84+ rule o Fluorescence recovery after photo bleaching (FRAP) technique  Fluorescent dye- labeled outer surface of cell  Shoot laser (light)  washes out fluorescence in a patch (bleached)  Watch how long it takes for molecule to move and replace bleached area  Recovery time is rapid  Bleach molecules diffuse out of region and unbleached molecules diffuse in  More cholesterol  more rigid  less rapid  Movement through membrane  Rate of recovery depends on diffusion coefficient  Fluorescence intensity vs time  Receptors respond to signal and move toward it lipids facilitate movement  easily broken  Movement occurs within layer lipid is found  Lateral diffusion- hydrophilic moiety  Traverse diffusion- (flip-flop) slower; rare CHAPTER 12 SUMMARY  Membranes o 60 to 100 angstroms thick o Proteins and lipids, covalent bonds o Highly selective barriers o Create compartments o Protein regulators  Fatty acids are key constituents of lipids o Hydrocarbon chains with carboxylic acid group o 14 to 24 C, saturated or unsaturated o Short chain length and unsaturation increase fluidity  lower melting temperature  3 common types of membrane lipids o Phospholipids, glycolipids (sugar), cholesterol (modulates membrane fluidity, steroid nuclease) o Amphipathic- 1 hydrophobic, 1 hydrophilic end  Phospholipids and glycolipids readily form bimolecular sheets in aqueous media o Hydrophobic interactions with fatty acid tails o Hydrophilic heads interact with aqueous medium o Lipid bilayers are cooperative; weak bonds  Impermeable to ions and most polar molecules but are fluid  Act as solvent for membrane proteins  Proteins carry out most membrane processes o Transport, communication, and energy transduction o Integral and peripheral proteins  Bound to membrane by electrostatic and hydrogen bond inducing beta strands o Sequence of 20 consecutive nonpolar AA  membrane spanning alpha helical region  Lipids and many membrane proteins diffuse rapidly in plane of membrane o Asymmetric o Ex: restriction of sugar residues on exterior surface o Dynamic structures- lateral and transverse diffusion o Degree of fluidity depends on chain length and saturation and cholesterol contents in animals  Eukaryotic cells contain compartments bounded by internal membranes o Ex: double membrane around nucleus (contains info) and mitochondria (produces ATP) o Receptor mediated endocytosis  enables intracellular vesicle formation when ligands bind to correct receptor proteins in plasma membrane o Fusion of vesicle to membrane  key step in release of signaling molecule outside cell CHAPTER 35: MOLECULAR MOTORS Actin and Myosin  Most molecular motor proteins are members of the P-Loop NTPase Superfamily-skeletal muscle  Cells in culture o Myoblasts  Not true stem cells  Still able to proliferate  Mature skeletal muscles cannot proliferate  Makes cell reproduction difficult/impossible  Can still grow (helper cells grow) o Beginning of mature muscle cell  Fuse together  multi nucleus cell  express genes  production of proteins  banded pattern o Myo D – add to culture, binds to receptor on surface  signal transduction  goes into nucleus and alters expression of genes  Genes associated with proliferation are off, making proteins on  Skeletal muscles o Nuclei on periphery o Proteins make up myofibrils o Made up of sarcomeres – contractile unit o g-Actin- structural protein in all eukaryotic cells o Globular polymerizes to form fibers  f-Actin o Myosin- mechanoenzyme; can convert forms of energy (bonds of ATP into work); part is also structural o I band has Z line in middle  Z line- where active filaments are anchored o H zone- just myosin  Myosin molecules linked together in middle of H zone o Sliding filament model  Filaments slide next to each other  Contracted- muscle contraction depends on motion of thin filaments (actin) relative to thick filaments (myosin); filaments do not change length  Actin and Myosin rd o Isolated actin and myosin put in buffer  pour together in 3 beaker  became move viscous  significant interaction between them o Viscosity could be reversed by adding ATP  o Add ADP  actin and myosin rejoin o Myosin molecule  2 regions  Region of protein that is globular  where enzymatic activity is  Region that is fibrous  no enzymatic activity  Myosin o Heavy meromyosin (HMM) migrates more slowly on gel o Neck region, separate globular domain from fibrous portion o Can cut with trypsin  cuts off tail o Globular domain – binds to actin  ATPase  signals conformational change o Fibrous domain  Alpha helical coiled helix; within primary sequence  7 AA repeat  Amino acid A and D (1&4) are hydrophobic; B, C, F hydrophilic o Hydrophobic A&D on same side  find each other; B, C, F face outward o Take single alpha helical region of neck and strengthen it  Actin binding site  P-loop  Nucleotide binding site  Essential light chain  Regulatory light chain o Thick filament  Wrap myosin around each other  Head region in both directions  bidirectional  Presence of myosin head domains at each end and narrow central region  Actin o Unidirectional o + and – end o Also an ATPase o Used to assemble into polymer  Interaction of thin and thick filaments o Optical trap  Actin filament stretched around 2 ceramic beads  suspended certain distance by laser beams  holds beads, measure polar stress  Supply ATP to system  Bind to globular domain of myosin  Hydrolyze  walk along  actin filament moves in relation to bead o Motor protein in action  ATP supplied  distance beads moved relative to laser beam  Pulling actin filament in step like process (beads can’t move)  1 ATP molecule hydrolyzed o Myosin binds well to actin with ADP in active site  ATP replaces ADP  Affinity for actin  myosin lets go of actin  Major conformation change between myosin globular domain and actin fibrous domain  90 degree shift  hydrolyze ATP to ADP  ADP in active site – affinity increases  Power stroke- propels myosin relative to actin or vice versa; conformational change back to original o Intermediate analog- freezes reaction (myosin-ADP-vanadate complex) o Position of lever arm when ADP is bound vs when ADP-vanadate is bound- 90 deg shift o P-loop  3 to 2 phosphates – small changes  transmitted to switches  then to relay helix  then to level arm  Control Actin-Myosin interaction o Tropomyosin- blocks binding sites o Troponin complex- wraps itself around actin filament; at each end of tropomyosin molecule  TnC- binds Ca2+  TnI- binds to actin  TnT- binds to tropomyosin o Muscle contraction- calcium regulated  Brain signals to move  calcium stored in SR  action potential to muscle leads to release of Calcium  calcium binds to TnC  tropomyosin moved  myosin can bind to actin  Ca pump  release actin  original position  TnC shifts  transmitted through subunits  moves tropomyosin Levels of complexity  Protein monomer  multisubunit (ex: dimer, trimer)  protein complexes (different subunits, ex: transcription)  ribosomes (assemble proteins)  organelles (ex: mitochondria- 100s of proteins, produces energy)  microtubules (scaffolding and communication)  Microtubules- form cytoskeleton o Structure prevents rapid change in shape o Connection between membrane and matrix o Railway system – motor proteins o Levels of cytoskeleton  Microfilaments- ex: actin; polymer of subunits brought together  Intermediate filaments- ex: keratin; dimers or trimers of alpha helical coiled helices  Microtubules- polymer, hollow cylinder, alpha and beta tubulin structures, bound to MTOC  Microtubule structure o Pulls chromosomes apart in cell division o Alternating alpha and beta tubulin; GTP at center of each o Helical fashion, hollow core o 1 end anchored to MTOC (microtubule organizing center)  Hydrolyze GTP to GDP  Falls apart o Dynamic instability- can form and take a part when needed Eukaryotic cilia/flagella o Microtubule arrangement 9 doublets, 2 singlets o Series of microtubules  axonene o Cell moves due to movement of flagella  Nexin- connects microtubules o Radial spoke- connects outer dimers to center o Motoprodyneine  ATPase  Acts like myosin with actin  Grabs tubulin, binds, hydrolyzes ATP  similar power stroke  releases  Motor proteins crawl down microtubules, bends microtubules  Dyneine- 6 active sites Kinesin  Tetramer- binding regions for tubulin on tail  Looks more like myosin  Uses microtubules to move  Moves molecules around cell (“train on railway”)  Carries neurotransmitters  2 motor domains (dimer), alpha helical coiled coil stalk, tail  Walks toward + end  As ATP is added to kinesin, displacement increases (walks further)  2 conformations o Kinesin-ATP complex  Neck linker- alpha helical region; in place of lever arm o Kinesin-ADP complex  ADP in active site of kinesin can still bind to tubulin but there are weak interactions, while ADP in active site of myosin cannot bind to actin o P-loop interacts with 3 phosphate  ADP  P-loop moves  switches relay helix  neck linker moves 90 degrees  How kinesin moves along tubulin o 2 heads in ADP form bind to microtubule o Release of ATP  conformational change – locks head to microtubule and pulls neck linker to head and throws 2 nddomain toward + end o ATP hydrolysis, 2nd head interacts with tubulin o Exchange of ATP for ADP in second head  pulls 1 head off tubule and st releases Pi and moves 1 domain along tubule o Cycle repeats Prokaryotic flagella  Tumbling- flagella not all together  Spin like a propeller to move  Counterclockwise- form super flagella  good propulsion  Clockwise- flagella spread out, move in different directions  Flagellin o Spinning motor o Energy source- proton motive force o Difference in pH  pH outside innermembrane is lower than pH inside cell (more H+ outside) o helical polymer of flagellin o flat surface facing core o hollow tube made up of multiple flagella subunits  Flagellar motor o Up to 40 distinct protein types o L ring- allows spinning without disruption o Motors in membrane o P ring- in peptidoglycan layer o Rod- spins (MS ring also spins) o H+ diffuse in o Motor components  MS ring- fliG subunits  Alpha helix  Motor proteins- don’t spin; pathway through which proteins move  Asp acid residue in middle  Motor proteins o Protons into outer channel o Transferred to MS ring o MS ring rotates counterclockwise o Protons released to inner half channel o Flagellum linked to MS ring  rotates with it o Outer half channel- proton in  fliG subunit Asp accepts H+ and moves and releases to inner half channel  proton transport coupled with rotation of flagellum  counterclockwise release of H+; causes spinning of MS ring and flagella  Course of E coli bacterium- tumble(clockwise, flagella untangle) counterclockwise- move straight Chemotaxis Signaling Pathway  Regulated by CheY  CheY bound to motor o When phosphorylated o Spins clockwise  tumble o Changes direction  Receptors in membrane initiate signaling pathway  CheY phosphorylation  CheY binds to flagellar motor  clockwise rotation  Attractant binds to receptor o Pathway blocked, counterclockwise rotation o Smooth swimming o CheY not phosphorylated, won’t bind, keeps motor moving in same direction  Repellant binds to receptor o Pathway stimulated, increased concentration of phosphorylated CheY, rotates clockwise o Tumble o Excessive phosphorylation changes direction to move away from repellent  Receptor protein unoccupied o A little bit of phosphorylation  clockwise  tumbling CHAPTER 35 SUMMARY  Most molecular motor proteins are members of the p-loop NTPase superfamily o Eukaryotic cells have 3 families of molecular motor proteins  Myosins  Kinesins  Dyneins o Proteins move along tracks defined by actin and microtubule cytoskeletons o Contribute to cell and ornaismal movement and intracellular transport of proteins, vesicles, and organells o Core P-loop structure- ability to change conformation in response to nucleoside triphosphate binding o Hydrolysis is key to motor function o Motor domains attached to extended structures that amplify conformational change and link core domains to one another or to other structures  Myosins move along actin filaments o Actin- 42-kd protein; polymerizes to form long filaments  Can bind either ATP or ADP o Myosin motor domain moves along actin filaments in cyclic manner o Begins with myosin bound to actin o ATP binds to myosin and is released from actin o Reversible conformational change occurs with hydrolysis of ATP while still bound to myosin  Lever arm moves o Myosin bound with ADP and P binds to actin o Pi is released from myosin which resets the position of the lever arm and moves actin relative to myosin o Release of ADP returns motor domain to original state o Length of lever arm determines size of step o Muscle contraction- sliding of actin (thin filaments) relative to myosin (thick filaments)  Regulated by tropomyosin and troponin complex  Prevent myosin from reacting until an increase in calcium concentration associated with a nerve impulse results in calcium induced changes in troponin and tropomyosin  Kinesin and Dynein move along microtubules o Microtubules- polymeric structures; alpha and beta tubulin  Similar guanine- nucleotide binding proteins (G-proteins) o 13 protofilaments with alternating alpha and beta tubulin subunits o Kinesins move with mechanism similar to that of myosin  Differences  ATP binding to kinesin favors motor domain binding rather than dissociation  Power stroke triggered by binding of ATP rather than release of P  Kinesin motion is processive- 2 heads work together and take turns binding and releasing microtubule; many steps taken before both heads dissociate; most move toward + end of tubule  A rotary motor drives bacterial motion o Rotating flagella for propulsion o Counterclockwise- flagella come together to form a bundle that propels effectively o Proton gradient across plasma membrane (not ATP hydrolysis) powers flagellar motor o Mechanism for coupling transmembrane proton transport to macromolecular rotation is similar to that of ATP synthase o Clockwise- flagella fly apart and bacterium tumbles o Bacteria swim preferentially toward chemoattractants – chemotaxis  When bacteria are simming in direction of increasing concentration of a chemoattractant, clockwise motion predominates, tumbling is suppressed  Biased random walk in direction of increasing [chemoattractant] CHAPTER 13: MEMBRANE CHANNELS AND PUMPS Membrane Permeability  Channels o Enables ions to flow through membrane in a thermodynamically favorable direction  Gradient which is established by pumps o Open for milliseconds  hundreds of thousands of ions flow through  Have selectivity  Don’t let through K+ or negative ions  Pumps o Establish gradients o Conformational changes  work against gradient o Build up gradient  requires energy; ATP, light, or other gradients o Opened and closed (regulated) by allosteric proteins Action Potential  Electrical signal  synapse  release of neurotransmitters from vesicles (transferred by kinesin) 10nm to 500 um  rapidly diffuse  bind to receptors on other side  Polarization disappears temporarily  ACH (acetylcholine) added  Depolarized postsynaptic membrane  Torpedo Marmorata – electric fish o Electric organ in head  emit voltage into water  shock attacker or stun prey o Used to treat people with gout and arthritis placebo affect ACH  ACH receptor o Pentramertric- 5 subunits  2 alpha that bind to ACH (ligand- small molecule that binds to a larger one) o Ligand gated receptor o Beta strands, 4 membrane spanning segments, alpha helices inside cell o Conformational change to open and close – lets + ions in  Action potential o Depolarization- increase membrane potential o Polarization- decrease membrane potential o Goes below resting potential  Patch Clamp Experiment o Taking patch of membrane of cell and clamping it to tip of pipet o Separates buffer outside clamp to inside o Apply electrode and see current moving through pipet o Whole cell mode  Suck membrane against tip of pipet  Burst and leave cell attached  Low resistance pathway between pipet and inner cell o Excised patch mode  Take out patch and get rid of rest of cell  Receptors inside pipet  Observing single channels o Measuring resistance (current) moving through pipet tip o Channel open  less resistance – spikes  ACH supplied, H+ ions in o Patch clamp experiment- 1 ion channel undergoing transition between open and closed states  At synapse o Channel closed- large hydrophobic side chains blocking middle o Rotation and sliding of helices o Channel open- small hydrophilic chains in middle (more polar) o Ach binds to alpha subunits  slight rotation of alpha subunits  transmitted to other subunits o Na out K in o Electrical selectivity- repels negatively charged ions o Enzyme to prevent permeable activity Voltage gated channels  Conductance change vs time- currents through Na and K channels underlying action potential; Na higher peak, K shorter and longer peak  Tetrodotoxin- neurotoxin produced by puffer fish  Large protein of 4 repeats of 6 alpha transmembrane helices  Voltage gated channel- not a ligand o Extremely polarized to depolarized  channel opens more freely  S4 helix responds the most to change in voltage o Glycine and arginine interact with adjacent negative charges to maintain position  rotates up  individual interactions change  change position  open channel (Na through)  Keep K+ from passing through- K bound to H2O is too large to pass through o Size limiting  Na channel vs Ca channel o Similar structure o K channel is ancestral; shaker  K+ channel in fruit flies o Tetramer o Mutation  Na channels work  K channels would respond to voltage but couldn’t shut off o K has to dehydrate to pass through channel  Thr or gly bind  Energy conservation of K+ o K aligns perfectly with side chains o Larger ionic radius o Lower energy state o Flows to other side ?


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