Class Note for BIOC 460 at UA 8
Class Note for BIOC 460 at UA 8
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Date Created: 02/06/15
BIOC 460 spring 2008 Lectures 8 and 9 Protein Func n Ligand Bind g and Allosteric Regulation Oxygen Binding to Myoglobin and Hemoglobin Allosteric Regulation of Hemoglobin Function Reading Berg Tymoczxo amp Stryer otn ed cnapter 7 pp isselgg problems in textboox cnapter 7 pp 2037204 34568 abbreviations used in tnis set of notes Hb nemoglobln Mb myoglobin Jmol structure of myoglobin littg vWWblmnem allzuna eduElasseSbluE4EZ4B2a le uglubm uglub ntml Jmol structure of hemoglobin littg vWWlllmnem allzuna eduElasseSbluE4EZ4B2a mulneniuglublnnewnb ntml Jmol structure of nemoglobin witn 237bl5pnospnoglycerate ZSVBPG bound lith MNWW blucnem arlmna editlassesblurAE24E2almulnbbggnewbgg lllml Key Concepts Ligand binding fundamentally important in biocnemical pnenomena Heme Fe protoporpnyrin lX in myoglobin and nemoglobin binds oz relersle witnout oxidation of tne neme Fe zvvnlcn is required for oz binding Myoglobln and nemoglobln s structures and ligand binding properties nave evolved differently for tne different functions of tne two proteins and tne structurefunction relationsnips are very well understood 7 Mb is monomeric l oz binding site per molecule nyperbolic binding curve no cooperativity 7 Ho istetrameric 4 oz binding sites per molecule sigmoid binding curve indicative of cooperative ligand binding structural communication between different binding sites by conformational cnanges 7 Ho is tnus an allosteric protein Key Concepts continued Hb is an allosteric protein 7 R state nxy cunfnrmallnn nign Ozblndlng affinity stabilized by oz binding oz is a nomotropic effector e T state deuxy cunfurmallun low oz binding affinity stabilized by binding of protons H 002 andor 23717lspnnspnuglycerale 237 BPG all neterotropic effectors allosteric innibitors e Allosteric regulation of oz bindingto Hb is important to ennance tne ability of HD to RELEASE oz in tne tissues ZSVBPG is needed in numan erytnrocytes red blood cells to reduce oz binding affinity enougn to get effective release of oz in tissues 7 ZSVBPG binds in central cavity of Hb stoicniometry BPSH17 tetramer Fetal Hb HbF nas different quaternary structure from adult HbA 1sz V5 Mb 7 Sequence difference between y and it reduces HbF s affinity for ZSVBPG tnus increasing its affinity for oz underpnysiological conditions Learning Objectives Terminology ligand fractional saturation prostnetic group cooperativity protomer binding site allosteric allosteric site allosteric effector allosteric regulation Briefly describe tne tertiary structure of myoglobin and tne nemoglobin subunits tne globln fold explain now tne nelices are designated andtne roles of tne proximal and distal HlS residues in neme and oxygen binding Write a general protelnellgand bindingdissociation reaction in botn tne association and dissociation directions Wnat istne matnematical relationsnip between tne association and dissociation equilibrium constants Describe now and wnere in tne structure of myoglobin and nemoglobin o2 binds including roles of protein functional grou s and neme and tne oxidation state of tne neme Fe required for o2 inding Sketch tne o2 binding curve v fractional saturation vs p02 for NONcooperative ligand binding to a protein sucn as tnat for o2 bindingto myoglobin On same plot slltetcn a binding curve tnat snows cooperativity cooperative ligand binding sucn as tnat for o2 binding to nemoglobin and explain again wnat is meant by cooperativity On botn curves indicate tne value of Ru tne po2 at wnicn fractional saturation of protein witn o2 is 0 5 ln wnat part of tne cooperative binding curve wnat part of tne ligand concentration range is protein predominantly in conformation witn low ligand binding affinity and in wnat part of tne ligand concentration range is tne predominant form tne nign binding affinity conformatlon Learning Objectives continued Explain now nemoglobin worllts pnysiologically in yiyo i e now cooperativity in 02 binding to nemoglobin facilitates loadlng of o2 in tne lungs and unloading of o2 in tne tissues lncludetne role of tne R state oxy conformation and tne T state deoxy conformation of nemoglobin Briefly describe tne structural cnan ge tnat occurs wnen o2 binds to tne neme of a subunit of nemoglobin including a wnat in tne neme structure triggers tne protein structural cnange wnen o2 binds b now tnat first protein structural cnange is communicated to otner subunits to cnange tne quaternary structure and tne o2 binding affinity of tne otner subunits and c effect of tne quaternary structural cnange on size of tne central cavity Explain tne effect of 23ebispnospn oglycerate on tne affinity of mammalian nemoglobin for oxygen and describe wnere on tne nemoglobin molecule 2378PG binds now many molecules of 2378PG bind to one nemoglobin tetramer and predominantly by wnst type of noncoyalent interactionstne 2378PG is bound Does 2378PG bind to tne R state or tne T state of nemoglobm Learning Objectives continued Explaln Why maternal red blood Cells release 02 and fetal red blood Cells bind 02 in tne placenta in terms of a tne difference in protein primary structure and quaternary structure subunit composition between HbA adult maternal and HbF fetal nemoglobin b tne effect of tne structure of HbF on its 2378PG binding affinity compared to HbAand c tne resultant difference in 02 binding properties of tne 2 nemoglobins and its pnysiological significance Discuss tne Bonr effect 0 binding and co2 blndlng in terms of a tne effect of increasing concentrations of eitner of tnese ligands on tne o2 binding curve for nemoglobln b tne pnysiological significance of tnis pnenomenon For a mutant ln wnlcn tneTrR equlllbrlum l5 snlftedtovvard the R State wnattype of cnange wouldyou expect in Pm Does tnat mean 02 affinity of mutant is nigner or lower tnan normal For a mutant ln wnlcn the TR equlllbrlum l5 snlfted toward the T State wnattype of cnange wouldyou expect in Pm Does tnat mean 02 affinity of mutant is nigner or lower tnan normal LEC 8 and 9 Protein Function ligand bind39ng and allosteric regulation of hemoglobin BIOC 460 spring 2008 Ligand Binding General case ligand binding to a protein I The ssenc 0f pT e funcnonlacnon ls BINDING reCOQnmon 0f chemical equation for dissociation of ligand L from protein P and Interaction With other molecules PL P L BINDING result of specific usually NONCOVALENT interadwns between m lecu39ar su39faces equilibrium dissociation constant Kd for reaction SHAPE complementarity lots of van der Waals interactions p L CHEMICAL complementarity hydrogen bonds salt linkages Kd PL HYDROPHOBIC EFFECT hydrophobic ligand minimizes 3 1 exposure to water by binding in hydrophobic site in protein What kinds of interactions give the most SPECIFICITYin binding Concentrations of free protein empty binding sites P free ligand L Terminology and PL complex PL in this expression are the equilibrium LIGAND a molecule or ion usually small that39s bound by concentrations another molecule usually large eg a protein COOPERATIVE ligand binding quotcooperativityquot binding of a ligand 02 to 1 binding site affects the properties binding affinities of other binding sites on other subunits of the same protein molecule Example 0 binding to Hb Kassociation 1 dissociation FRACTIONAL SATURATION L Fractional Saturation Y Fractional saturation Y fraction of total binding Sites on protein Kd L 31W W M occupied sites equation for a rectangular hyperbola Y occupied binding sites I total binding sites units of Y P L P L o a 5 es minimum and maximum values of Y lt Plot of Y vs ligand Binding ofa ligand leuunu PL 0 apmte39quot Photal P P39L oKd concentration of 10 ligand needed to Kd P L HALFSATURATE Y M P39L eq the binding sites L o 00 WhenY 05 L Kd DKd 1o 20 3o Ku L Suppose you have 2 proteins that both bind L Plot of fractional saturation Y vs ligand Is hyperbolic the same ligand one with Kd 10 5 M and the Approach to site saturation is asymptotic therWith Kd 10 7 M Which one has the higher binding af nity for the ligand Myoglobin Mb Myoglobin REVIEW from tertiary structure structure Jmol structure of Mb binds 02 in muscle cells for storage and for intracellular transport using a heme group mostly 70 a helical rest mostly turns amp loops at surface first highresolution crystal structure of a protein ever determined very compact structure almost no empty space inside very watersoluble 5 Pro residues 4 in turns 8 or helices designated by letters A H from N to C Berg et al Fig 2488 heme black with purple Fe2 termmus Nelson amp Cox Lehni39nger Principles of Helices amphipathic surface sides hydrophrlrc R groups Biochemistry Fig 416 buried sides more hydrophobic R groups 5 heme in red blue residues LeU Ile Val Phe LEC 8 and 9 Protein Function ligand binding and allosteric regulation of hemoglobin BIOC 460 spring 2008 Myoglobin structure continued 02 binds to heme prosthetic group of Mb and Hb Distribution of Amino Acids in Mb structure 39 m iron pr t p rphyrin IX With bound Fe2 h d h b d H h d d N m m 1 heme per Mb 1 OZ binding site per Mb molecule y mp o I res39 ues m ye OW c alige res39 ues m ue 9539 W 39e 1 heme per Hb subunit so maximum OH 02 can bind to Hb tetramer A surface View B crosssectional View showrng interior of protein NOTE many charged residues on surface none in interior a many hydrophobic residues in interior but also a few on surface The only polar residues inside are 2 His residues involved in binding the heme and OZ Propionate side chain 6 coordination positions of heme Methyl group i CH3 K HZC Vinyl group I CH3 e HCH2 fepruloporphyrin IX Nelson amp Cox Lehninger Principles Berg et al Fig 249 Berg et al p184 OIBiochemistry 4th ed Fig 51 Heme Fe2 coordination o2 binding by myoglobin 4 positions gt to 4 N atoms in heme all close to being in same plane Myoglobin monomeric single polypeptide chain H H H just 1 O2 binding site per molecule fgs i39elji etgrgtleri na glitz gl e lix Mb39s OZ binding noncooperative no communication is possible p between different binding sites because each site is on a different His F8 the proxrmal His L hydrogen bond to molecule 6th coordination position is Hiszg39gal O atom Mboz Mb 02 to 02 when 02 binds O2 binds between Fe2 Phe cm i K Mb 02 and anotherHis in protein 9 VIIEHQ r d 39 ii 3021 HisE7 the quotdistal Hisquot 2 hydrophobic residues Val Y Ozibound Mb3902 and Phe help keep Fe2 from Mbhotal Mb Mb02 becoming oxidized to Fe n 39 IHis 2 p 2 Binding of O2 to myoglobin showmg H F me39ma coordination of one 0 to the Fe2 395 B F 39ggefj tw me Kd 02 P50 p02 of the heme and hydrogenbond of Fey so if Fez I PI t fY o h b I the otherOto distal His His E7 moves F helix 0 0 VS P 2 395 W79quot 0399 Nelson amp Cox Lehninger C mOVeS Principlesdsmhemsw 4th ed Fig 556 P50 is formally equivalent to Kd the ligand conc p02 when Y 05 Plot of fractional saturation Y vs p02 for Mb HemOQIObin a hetemtet ame 01232 W 02 binding by myoglobin OZ transport protein p02 02 concentration in Berg 9 Fig 7396 very wellunderstood example of allosteric regulation important concept in regulation of activity of many enzymes as well O2 binding to hemoglobin is cooperative Biochemical allosteric control understood at level ofmolecular structure related to physiology of whole organism pressure units torr O 1torr 1 mm Hg at 0 C and standard gravity i e sea level 2 identical or subunits red structurally similar to 2 identical B subunits yellow on and 5 also very similar to structure of myoglobin both Pu2 2 torr P50 primary and tertiary structure gene duplication of single P5 ligand 02 cone in pressure units when Y 05 50 saturation Y fractional saturation P U Mb function 02 storage and 39 39 39 39 ancestral ene and subse uent mzssgrwrin39skzrlirstream o 25 so vs mo of q requires no regulation p02 torr sequences gt different globin genes Suppose a mutant myoglobin has a P50 of 5torr ls its 02 binding tertiary quotfoldquot globin foldquot r 39 tighteror weaker than that of normal Mb shown on graph conserved through evolution Berg 9t alr Fig 254 LEC 8 and 9 Protein Function ligand binding and allosteric regulation of hemoglobin BIOC 4 60 spring 2008 Tetrameric Quaternary Structure of Hb 11232 132 Hemoglobin39s chains spontaneously assemble into quaternary structure Quaternary structure stabilized by noncovalent bonds no disulfide bonds in Mb or Hb Hb structure a quotdimerquot of 2 up protomers 192 T ai l 1252 Each on has one 5 quotpartnerquot with which it is more closely associated Conformational changes in tetramer affect Hb39s affinity for 02 B i 39 Bergetal V Fig75 Binding of 02 to Hb is sigmoid not hyperbolic Sigmoid binding curve Y vs ligand Lo Myoglobin Indicates that there are 3 multiple interacting 2 binding sites for ligand 1 08 Hemoglobln on each protein molecule 5 the 4 different sites 0 6 communicate with each a 39 other cooperative 3 binding allosteric 04 effects 2 5 Mb has 1 02 binding site 2 02 per molecule so no 395 opportunity for interaction gt39 00 no communication 0 25 50 75 100 between binding sites hyperbolic binding P02 torr curve noncooperative binding Berg etal Fig 77 Oxygen binding Hemoglobin Allosteric protein Greek allos other shape def Binding of ligand to one site on multisubunit protein affects the binding properties of another site on same protein molecule stereos Hb 4 02 sites per Hbtetramer sigmoid binding curve diagnostic of cooperative binding cooperativity communication between different ligand binding sites on same multimeric protein molecule Communication occurs via structural conformational changes 2 interconvertible conformational states of Hb T state and R state Physiological reason for regulation of O2 binding of Hb Why would Hb want to regulate the tightness of its 02 binding such that because of a conformational change the protein binds 02 with higher affinity tighter binding at higher 02 concentrations lower affinity weaker binding at lower 02 concentrations Explana ion lies in 02 transport function obe o 2 aspects of transport a binding 02 in lungs b releasing 02 in rest of tissues Cooperativity enhances O2 deliveryrelease by Hb LEC 8 bindin and 9 Protein Function ligand 9 and allosteric regulation of hemoglobin The sigmoid 02 binding curve of Hb a composite Low af nity curve at low 01 conc T state predominating High af nity curve at high 02 conc R state predominating T state low 02 affinity ltgt R state high 02 affinity n absence of Oz equilibrium lies far toward T state weak 0 binding In presence of Oz equilibrium is shifted toward R state tight binding Rstate b39 d39 gcurve O2 binding is 08 a molecular switch that induces a 06 Observed hemoglobin binding curve change in 02 Y fractional saturation binding affinity 04 over the whole population of 02 Hb molecules quot Tstate binding curve in solution on 0 50 100 150 200 p02 ltorr 5397quot Cooperativity enhances O2 deliveryrelease by Hb Sigmoid curve Hb is 98 saturated with O2 in the lungs where p02 100 torrs At high 02 conc both Mb and Hb are essentially saturated with 02 Difference is clear at Tlssues Lungs Myoglobin lower 02 conc in 10 tissues where Mb Hemoglobin stays loaded doesn t o8 unload much 02 No With a sigmoid curve 0396 Cooperativity Hb can UNLOAD release dissociate more ofits carrying capacity of Oz in the tissues where pO2 20 torr than hypothetical P 4 Y fractional saturation P N l it could release with 00 noncooperative 0 2 50 100 150 200 binding Berg et al Fig 77 P02 3 BIOC 460 spring 2008 O T V Paria in 02 3 Iron Structural basis for cooperativity in Hb OZ binding changes position of Fe in heme of Hb initiating structural changes In absence of bound OZ heme iron lies slightly outside porphyrin plane bound coordinated to an N of a His residue the proximal His His F8 When OZ binds Fe moves into plane ofheme pulling with it His F8 residue 0 07 0 J In oxyhemoglohin In deoxyhemoglobin Tertiary structural changes in T gt R 02 binding pulls proximal His F8 toward heme moving F helix Conformational change in one subunit causes structural changes in interface between al l and 9131 protomers eventually triggering quaternary change in whole Hb tetramer a v 00 0 39 mp a anti 09 30 OxyHb red deoxyHb gray alli rr2 pz interface Deoxyhemoglobin Oxyhemoglobin Berg et al Fig 714 Quaternary structural changes in T gt R al l protomer shifts relative to clip protomer and rotates 15 Figure is looking down 2fold symmetry axis through central cavity subunits yellow Note decrease in size of central cavity in R state compared to T 15 Deoxyhemoglohin nyhomoglobin Berg et al Fig 710 Animations showing T gt R conformational changes as 02 binds R state has red O bound Overall change summary Note change in size ofcentral cavity Fe moves into plane ofheme when OZ binds htthMMnAbiochemarizonaeduclassesbioc462462aNOTEShemoglobinoxylhtml Proximal His moves with iron so F h 39 39 39 l 39 elix moves mm Movement of F helix alters tertiary structure of that individual subunit htthMMMbiochemarizonaeduclassesbioc462462aINOTEShemoglobinoxy3html Tertiary changes in individual subunits cause structural changes in protomer interfaces between 12 and it and between at and p1 htthMMMbiochemarizonaeduclassesbioc462462aINOTEShemoglobinoxy4html When at least 1 OZ has bound to each 045 protomer so at least 1 subunit per protomer has changed tertiary conformation whole quaternary structure shi s httijMwbiochemarizonaeduclassesbioc462462aNOTEShemoglobinoxy5html Any condition that shifts the R T T equilibrium toward the R state increases the O2 binding af Any condition that shifts the R 3 T eqUIlIbrium toward the T state decreases the O2 binding af nity 3 allosteric inhibitors of O2 binding quottunequot the O2 affinity of hemoglobin 23bisphosphoglycerate 23BPG protons H carbon dioxide C02 Why are allosteric inhibitors of O2 binding needed Purified human Hb much higher 0 binding affinity than Hb in erythrocytes red blood cells Without some negative allosteric regulator to reduce its af nity for OZ human Hb wouldn39t be able to unload much OZ at all in the tissues Hb would release only about 8 of its payload at 20 torr 23bisphosphoglycerate 23BPG main allosteric inhibitor of O2 binding to human Hb 23BPG metabolic quotbyproductquot produced by isomerization of glycolytic intermediate 1 3BPG in red blood cells highly anionic structure glycerol CHZOH CHOH CHZOH glyceric acid propionic acid a 30 carboxylic acid with OH groups on C2 and C3 0 0 C2 and C3 OH groups esterified to phosphates in 23 BPG 239339B39sph sph 9ly39 quot39e 23BPG Where In Hb structure does 23 BPG bind By what type of interactions How does it reduce the O2 binding af nity of Hb LEC 8 and 9 Protein Function ligand binding and allosteric regulation of hemoglobin BIOC 460 spring 2008 Berg Fig 716 23 BPG binds to p chain residues in central cavity of Hb tetramer Jmol structure of BPGHb httglwwwbiochemarizonaedulclassesbiout62462almolhbbggnewbgghtml stoichimetry of 23BPG binding 1 BPG per Hb tetramer 23 BPG does NOT bind where the O2 binds 3 charged groups from each 5 chain in central cavity help bind 23 BPG by ionic interactions N 9 B1 subunit g a U C o l 9 3 His 9 0L 32 9 vs i 09 a a 0 3 00 a 0 33 His143 His1 c et al What would be the effect of losing a charged group from BPG binding site and howwould that affect O2 binding af nity Fetal Hemoglobin pregnant woman 02 taken in by mother through her lungs is transported by maternal adult Hb a2 2 to placenta for deliveryto fetus ln placenta maternal adult Hb must release 02 and fetal Hb must bind 02 For effective transfer fetal Hb must be able to bind 02 more tightly than maternal Hb How does fetal Hb manage to bind O2 tighter than maternal Hb Different globin genes expressed at different times in embryonic development encoding different Hb subunits with OZ binding properties tailored to embryo39s needs at that stage Last 23 offetal life predominant form of Hb present is 0272 y chains are being made rather than 5 chains 3 vs y similar AA sequences but crucial differences chains have His 143 in 23BPG binding site y chains have Ser 143 in 23BPG binding site like Berg et al Fig 710 but in ribbon diagram Central cavity ofT state of Hb the deoxy conformational state big enough for 23BPG to t Quaternary structural change from T to R state shrinks central cavity not enough room for 23BPG to bind in R state s central cavity 23BPG binds only to T state stabilizing T state and shifting equilibrium toward T weak OZ binding form and away from R so whole sigmoid O2 binding curve is shifted to higher 02 concentrations weaker O2 binding higher P50 15quot L T state deoxyHb R stale oxyHb Fetal Hemoglobin continued A lower fraction of Hb molecules with 23BPG bound means more of fetal Hb is in R state more than maternal Hb Thus under physiological conditions at conc of23BPG found in erythrocytes fetal Hb has a higher 02 binding af nity than maternal Hb Thus mother can quotdeliverquot 02 to fetus o2 af nity of fetal 10 red blood cells Fetal red Maternal red cells 53 co 9 1 Fetal Hb binds 02 more tightly than maternal adult Hb because fetal Hb binds 23BPG less tightly than adult Hb does 5 02 flows from maternal oxyhemoglobin to fetal deoxyhemoglobin N Y fractional saturation P o 0 50 100 Berg et al p02 torr Fig 717 Other negative regulators of 02 binding to Hb H and C02 Binding of protons and binding of 002 promote release of 02 weaker binding ofoz Protons and 002 preferentially bind to the T state of Hb shift T 4 R equilibrium toward T state reduce 02 binding affinity of Hb Effect of H and C02 to promote release of 02 from Hb the quotBohr effectquot Why is it useful physiologically that protons and C02 bind more tightly to T state than to R state Physiological role of H and C02 as negative allosteric effectors of 02 binding to hemoglobin Lungs Hb quotwantsquot to bind OZ tightly to quotload upquot pH is quothighquot pH 74 H is low 002 is low because it39s being gotten rid of exhaled 02 is high Ligand conc conditions all favor R state Result 02 binds tightly That39s what you want to BIND 02 maximal quotloadingquot in the lungs Tissues Hb quotwantsquot to dissociate its 02 to UNLOAD pH is quotlowquot pH 72 H is high because catabolism breakdown of nutrients produces protons acid especially lactic acid in active muscle tissue 002 is high because COZ end product of oxidation of C atoms in catabolism of nutrients 02 is low Ligand concentration conditions all favor T state Result 02 binds weakly That39s what you want to DISSOCIATE 02 maximal quotunloadingquot LEC 8 and 9 Protein Function ligand binding and allosteric regulation of hemoglobin BIOC 460 spring 2008 Effect of pH and of CO2 on O2 binding affinity of Hb the Bohr effect Chemical basis of Bohr effect Tissues at least partially understood 10 02 binds to 1 amino groups of T state Lungs 08 Proton concentration some groups have different pKa values in R state from their pKa values in T state higher pKas in T state Higher pKa in T state means that ionizable group binds protons more tightly in T o 0 state than in R state 39 06 04 02 Y fractional saturation In the T state when those groups are protonated they can form p02 or salt links with charged groups that can t form in R state PH 73941quot COZ Those salt links stabilize T P 72 02 599 e 5 conformation pH 7240 torr C02 Fig 7721 Mutant human hemoglobins Mutant hemoglobins provide unique opportunities to probe structure function relations in a protein There are nearly 500 known mutant hemoglobins and gt95 represent single amino acid substitutions About 5 of the population carries a variant hemoglobin Some mutant hemoglobins cause serious illness The structure of hemoglobin is so delicately balanced that small changes can render the mutant protein nonfunctional 4 types of mutant hemoglobins Properties are altered in one of the following ways 1 Mutation in heme binding pocket leads to loss of heme 2 Mutation disrupts tertiary structure of a subunit 3 Mutation stabilizes methemoglobin Fe oxidation state of heme in Hb 4 Mutation stabilizes the R state or stabilizes the T state compared to their stabilities in normal HbA 1 4 types of mutant hemoglobins Mutation in heme binding pocket leads to loss of heme produces a nonfunctional protein can39t bind 02 2 Mutation disrupts tertiary structure of a subunit produces protein with reduced stability or impaired function or both 3 Mutation stabilizes methemoglobin Fe oxidation state of heme in Hb In order for hemoglobin to reversibly transport 02 iron must remain in ferrous Fe2 state Oxidizing iron to Fe3 produces metHb which does not transport 02 Red blood cells contains enzymes that can re reduce the iron in the occasional normal HbA molecule whose iron gets oxidized Mutations that stabilize metHb provide a negatively charged oxygen atom as a ligand for the iron eg Glu instead ofthe normal distal His imidazole N Negatively charged oxygen ligand stabilizes iron in the Fe3 state Mutant hemoglobins continued 4 Mutation stabilizes the R state or stabilizes the T state compared to their stabilities in normal HbA Mutations at the subunit interfaces between the two up protomers o en interfere with quaternary structure ofhemoglobin Such mutations can change the relative stabilities of hemoglobin39s R and T states shifting the equilibrium more toward R or more toward T state thereby affecting O2 af nity of mutant hemoglobin Normal HbA has P5 26 mm Hg 26 torr or about 35 kPa P50 is the p02 at which the fractional saturation 05 so half of Hb39s 02 binding sites are occupied For a mutant in which the TR equilibrium is shifted toward the R state what type of change would you expect in P50 Does that mean 02 af nity of mutant is higher or Iowerthan normal Would the P50 for the mutant be higher or lower than normal For a mutant in which the TR equilibrium is shifted toward the T state what type of change would you expect in P50 Does that mean 02 af nity of mutant is higher or Iowerthan normal Would the P50 for the mutant be higher or lower than normal LEC 8 and 9 Protein Function ligand binding and allosteric regulation of hemoglobin
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