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Lecture Material for Test 5.docx

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Lovaye Kajiura

Lecture Material for Test 5 November 10, 2010 Chapter 7 Slide 1 -myoglobin in our muscle to facilitate diffusion of oxygen into muscles -first found in sperm whale as an oxygen storing molecules -monomer, single polypeptide chain, 8 alpha helices, globular -heme molecule is site where iron II (ferrous) is located -ferric iron insoluble, ferrous iron is reduced form -smaller than hemoglobin thus was solved first -hemoglobin and myoglobin are homologous -hemoglobin is a tetramer -more complex to solve, took more time to solve -soak crystals with mercury -can change intensity of amplitude -structure is not compromised by heavy atom -change in amplitude allowed calculation of phase -phase and amplitude together allowed solving of electron density which then can be used to solve structure Structure of sperm whale myoglobin -8 alphahelical chains -Kendel created his own nomenclature for chain and atom identification -still used today for myolgobin -modified for other structures -heme group bound to helice c and F in myglobin The heme group -tetra pyrrole – creates pii electron system which absorbs electromagnetic energy -gives myoglobin its colour -binds iron -histidine residue that is important for ligating heme group -ferrous II can be converted to ferrous III in presence of oxygen -blood turns brown with oxidated to ferrous III -groups in close association to place tetra pyrrole ring in right plane -onces oxygen ligated to 6 position, oxygen hydrogen bonded to another side chain of histidine -positions the iron so it can bind optimally to oxygen -fraction of myoglobin that is oxygenated: fractional saturation -when it at ½ = useful constant: related to eq’m constant -concentration of oxygen refers to partial pressure -p50 – molecule is half saturated = K -rectangular parabola -binds in 1:1 ratio since it’s a monomer Hemoglobin structure -alpha and beta structures are homologous to each other -conformational change when iron binds First experimental evidence of an allosteric change in protein Oxygen Binding Curve of Hemoglobin -sigmoidal curve -uptake is cooperative November 15, 2010 Structure of sperm whale myoglobin -solved by John Kendrew -8 alpha helical segments -soluble globular protein -tetrapyrolle group where oxygen binds (to iron) -myoglobin in whale stores oxygen for diving -in humans, used for transporting oxygen from blood to peripheral tissues (skip a few slides) Oxygen –binding curve of myoglobin -1:1 ratio -as partial pressure increases, saturation increases -at p50 : partial pressure of o2 when saturated at 50% Hemoglobin structure a)deoxyhemoglobin -hemo 10 times less affinity of myoglobin -when one oxygen binds, the rest of are induced to bind -result of having similar/same parts in a protein -tetramer affords it a mechanism to sense the oxygen concentration and change the affinity for the remaining unit: cooperative process: binding to one that induces conformational change that is communicated from one subunit to another subunit -get sigmoidal relationship b) hemoglobin -a little diff than deoxygenated -grey arrows show slight twisting First experimental evidence of an allosteric change in a protein -known that crystals of hemoglobin had a diff structure -crystallographers had to solve the structure twice from crystals since deoxy and oxy were diff -crystals formed in soln -created anaerobic environment: deoxy -pump nitrogen into soln -fig 7-6 -shows subtle change in beta and alpha subunits Oxygen-binding curve for hemoglobin -myoglobin has rectangular hyperbola saturation curve (1 oxygen per myoglobin -monomer so can’t change another myoglobin -sharper: has higher affinity -dotted curve: if hemoglobin binding wasn’t cooperative -hill eqn -theoretical binding -can get much or saturation at p50 where physiology is needed -as hemo moves through circulation, gives out oxygen -myo needs a a lower pressure to become saturated since it is exposed to much lower concentrations of oxygen -fractional saturation at 50% gives p50 Mathematical relationship -Hill eqn -n = number of subunits -assumes that it binds simultaneously though that is not really happening -use it to determine the degree of cooperative binding -hem 2.8 to 3 -if n = 4 = infinite binding Fig 7.8 -adjust the value of n until it fits the experimental data -btw o.1 and 0.9 = straight line relationship -slope of hill plot where saturation = half to give coefficient -hemo has around n of 3 -value is less than 4 since cooperativity is not infinite -but since it is greater than 1 , cooperativity if quite high -oxygen binds with a 100 times greater affinity -eq’m constant (p50) is lower when affinity is higher -small K = high affinity T state and R state -this is not unique to hembolobin -T = tense -R = relax -deoxy seemed to be more tight -oxygenated tend to more relaxed Movement of the heme and the F helix -there is a diff -when oxygen binds , satisfies electronic requiremtns of iron -as covalent bond forms betw oxygen and Fe -Fe binds less tightly to heme group -doesn’t have the same requirements -thus relaxes -causes porphoirin ring to lose distortion -moves to a a planar state Which causese heme group to be dragged down -to accommodate small change, helix shifts -altered by less than 1 A -helix tilts , larger change at ends of helix Changes at the alpha-beta subunits interfaces -aa at interface btw the alpha and beta subunit to shift into a diff conformations -5 A shift -quanternary change -network of H bonding -series of ion pairing that occurs at the termini -alpha is positively charged -ion pairings manifested in deoxyhemoblobin -when it binds to oxygen, these interactions are torn apart -local environment of ionizable groups usually has a pH that can alter the pKa of the groups -as ion pairing that occur at termini are altered, pKa increases, has higher affinity for proton The Bohr Effect -when protein in lungs, partial pressure of oxygen is such that the protein picks up oxygen from the lungs -exercise – deplete amt of oxygen needed by muscle -muscle uses glycolysis, not citric acid cycle -lactic acid accumulates and pH starts to drop -when pH drops, the affinity of oxygen for hemoglobin starts to drop as well -hemoglobin when reaches peripheral tissues releases a greater amt of oxygen than before Figure 7.13 -carbon dioxide becomes soluble (Reacts with water to form bicarbonate and a proton) -rxn occur spontaneously but doesn’t occur fast enough -red blood cells also carry carbonic anhydrase that catalyses this rxn and makes it occur -one of the highest enzymatic rxns known -eq’m can be drawn toward bicarbonate direction by removing proton -hemoglobin can mop up proton due to increased pKa of conformational change -when it binds oxygen, pKa drops, proton released which drives the next rxn -bicarboante converted back to carbon dioxide and carbon dioxide is released The effects of BPG and CO2 on hemoglobin’s o2 dissociation curve -purified hemoglobin has higher affinity for oxygen than in blood -compound called BPG present in red blood cells that binds to hemoglobin -cuases conformation of T state to be stablilized -shifts p50 -also need CO2 which can rxn with amino terminus BPG -metabolite proceed in RBC that accumulates to 4 millimolar -binds to hole in tetramer -deoxy (T state) has big hole that narrows when oxygen binds -that site is structure to bind BPG -the negative charges on BPG are satisfied by positive on alpha and beta aa -that hole is only big enough to accomodate BPG in T state -BPG keeps hemoglobin in conformation in t state -beta subunit in fetus has a paralog -zeta subunit: his replaced by serine -positive charge replaced by neutral aa -reduced BPG affinity -fetal hemoglobin has higher affinity for oxygen than maternal hemoglobin -oxygen transfers to baby -high altitude training/adaptation -RBC increase (takes long time – about a week) -body can change BPG quite rapidly -BPG doubles – T state favoured -releases larger fraction of oxygen -degree of saturation does not improve but the release changes The symmetry model of allosterism -induced fit model – when protein bind to enzyme, conformational change -more like hand-glove model than lock-key model -allosteric control model (allo – another, steri - site or structure) -binding of ligand at one site causes a conformation change at another site -can only explain positive cooperativity (infinite cooperativety) The sequential model of allosterism -systematic – where one subunit binds, induces conformational change -may have higher of lower affinity -more general model where can explain both negative and positive cooperativity -these two models are general principles in multisubunit enzymes that involved in regulatory processes Table 7-1 -mutants can change p50 or degree of coopertivity -list of diff mutations Sickle cell anemia -provides a degree of resistance to malaria -that is why mutation is fixed in popn (African) -valine cause interaction btw hemoglobin molecules -an aggregate of hemoglobin Structure of a deoxyhemoglobin S fibre -glu to val causes a change in physical properties of molecule -causes it to aggregate -red blood cells have bi concave structure needed to travel through capillaries -sickle cells get clogged in capillaries and cuts of blood flow, eventually lethal Figure 7.20 Hydroxyurea -reduces sickling process, reduced blockage in capillaries and relieves symptoms -hydroxyurea induces expression of fetal hemoglobin -changes T to R eqm slightly -aggregation only occurs in T form (deoxygenated form) -increases oxygenated form – reduces aggregation Fig 7.21 -heterozygotes have -spleen removes old red cells in blood -cells that are infect with parasite of malaria -pH of envionrment drops -shif
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