Sp Top Bio Mechanism
Sp Top Bio Mechanism CHM 6304
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This 52 page Class Notes was uploaded by Brigitte Wyman on Friday September 18, 2015. The Class Notes belongs to CHM 6304 at University of Florida taught by Gail Fanucci in Fall. Since its upload, it has received 35 views. For similar materials see /class/207011/chm-6304-university-of-florida in Chemistry at University of Florida.
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Date Created: 09/18/15
Big Question We can see rafts in Model Membranes GUVs or Supported Lipid Bilayers LM but how to study in cells Do rafts really exist in cells Are they static large structures Are they small transient structures FRET and FRET based Microscopy Techniques 4 basic rules of fluorescence for overview presentation The FrankCondon Principle the nuclei are stationary during the electronic transitions and so excitation occurs to vibrationally excited electronic states Emission occurs from the lowest vibrational level of the lowest excited singlet state because relaxation from the excited vibrational energy levels is faster than emission The Stokes Shift emission is always of lower energy than absorption due to nuclear relaxation in the excited state The mirror image rule emission spectra are mirror images of the lowest energy absorption Fluorescence Jab03978 Diagram mm mu m n S excited rotational shines not shown A Dnomn absormlon F 7 fluorescence smlsslon no no escence r lslaie la 9 Stokes shift is the difference in wavelength or freguency units between positions ofthe band maxima ofthe absorgtion and luminescence sgectra ofthe me electronic transition en a molecule or quot quot it enters an excited electronic state The tokes shi occurs because the molecule loses a small amount ofthe absorbed energy before rereleasing the rest ofthe smkes 5m energy as luminescence This energy is H E hquot hCA rgy so electronic ground state intensity olten lost as thermal ene absorptmn wavelengm Icrnucluv distance 01 HIL mound snug Figure 112 nllllum r m slulc inmnd hl mpm Emlwun 1n 1 soc Hcfnrc phosphorus ncc MIL 1U39 52 Nunradialivc mt In c Imcmul conv zv mn A w Lwc Ahmrpuun Nnnmdmm c W 5m livlurc unwwuncc llr scc Emusmn IUquot gtL nd sungch I Iqu FrankCondon Principle and LeonardJones Potential hm Cwilmi mn Hrmnm sum Tulal E Mia m Factors Governing Fluorescence Intensity 1 Internal conversion non radiative loss via collisions with solvent or dissipation through internal vibrations In general this mechanism is dependent upon temperature As T increases the rate of internal conversion increases and as a result fluorescence intensity will decrease 2 Quenching interaction with solute molecules capable of quenching excited state can be various mechanisms 02 and l are examples of effective quenchers 3 lntersystem Crossing to Triplet State Quantum Yield number of photons emittednumber of photons absorbed Quenching Figure 151 Energylevel diagram ol39two chromophores G and SI indicate the ground and lirst excited states respectively heavy lines The vibrational levels are the thin lines A This molecule is capable of lluorescing by the transitiun solid arrow indicated in the diagram Alter excitation there are vibrational losses wavy arrow to the lowest level of the excited state and then emission from this stale dashed arrow B This molecule fails to uoresce because the vibrational levels ol G are higher than the lowest level olS hence there can he a nonradiative transition horizontal wavy arrows l mm SI to a vibrational level off followed y nonradiative losses to the bottom ol G vertical wavy arrow Common Fluorescence Applications in Biophysics Tryptophan Fluorescence Protein FoldingBinding Isotherms Fluorescence Quenching Protein Structure and Dynamics Fluorescence Anisotropy Binding Fluorescence Resonance Energy Transfer Binding Distances Conformations Table 82 Fluorescence Cl39iul39uclcrislim of protein and nucleic acid constilucms and cocnzymcs Mimipliva l lum usccnucquot Scmnwity Am nun Am r LiiiA0quot Substance fundiiions mm x I0quot nml my user x ll 5 Tryplophan H10 pll 7 80 6 343 020 26 11 Tyrosine H 10 pH 7 274 l 4 303 0 Id 16 20 Phenylalanine 0 pH 7 2 7 0 2 282 004 64 008 Yeast RNP39k K20 I quotl 460 007 63 091 Adeninc Hg 17 7 260 H 4 32l 26 x 10 quot ltl02 0032 Guanine 10 pH 7 275 X 1 329 30 X 10 4 lt002 0024 Cyiosim 0 pH 7 267 6 1 3 0 x I0 1 lt002 0005 Uracil H20 pH 7 260 J 5 308 04 x 0 4 lt002 0004 NADH H10 pH 7 Nil 6 7 470 009 040 12 Common Fluorescent Probes H30 CH CH12NHiCO CHII N I 1 12 6 V ansy chlmkle LSLAEDANS z I N N J gt KN I l Rmose FA hcnmldcmm no NH7CONH NH1 Pro klvmc molmscmiuurha7idc Ellndimn hmmlklc Flgu e 516 svrm rum Hf Her m pmmw lmud m Tuhlc 33 Tabla 83 Typical uorescent probes bsurption Emissiun Sensitiwty 11m Ema 11m Kr 39imml r Probe Uses lnml x 1039 hint ID nsuc x 10 3 Dunsyl chloride Covalent attachment to 330 34 510 01 13 34 protein ys s l5IAEDANS Covalent attachment to 360 6 8 480 05 15 34 protein Lys Cys Fluoresccin Covalent attachment to 495 42 516 03 4 l 16 isothiocyanatc FITC 39 8ltAnilinoilnaphthalcne Noncovalent binding to 374 78 454 098 16 67 sulfonalc ANS proteins Pyrencv and various Polarization studies on 342 40 383 025 100 100 derivatives large sys ems Ethcnmdcnosine and Analogs of nucleotides 300 26 4l0 040 26 10 various derivatives bind to proteins incorporate into nucleic acids Ethidium bromide Noncovalcnt binding to 515 3 8 600 1 265 38 nucleic acit s Pro avine Covalent attachment to 445 15 516 002 7 30 monnscmicurbazidc 39ends vmm Imwn M 4 ml l m m mmmun unnllcl xiluc nltcn Ilc I39mlnd Suucluru oflllcsc wuhcs an shown in l tgum tllb mhm lcmpumltllc Omar considerably Sensitivity to Local Environment luorescence can be used to probe local environment because of the relatively long lived singlet excited state 10399 to 1039E sec various molecular processes can occur Protonationdeprotonation Solvent cage reorganization Local conformational changes Translationslrotations example a intensity and wavelength of uorescence can change upon going from an aqueous to nonpolar environment This is useful for monitoring conformational changes or membrane binding b Accesibility of quenchers location on surface interior bilayer etc FRET Fluorescence Resonance Energy Transfer DA2DA Sensitive to interactions from 10 to 100A Increase acceptor sensitivity Quenches donor fluorescence Decreases donor lifetime E k1Jkrrkl kn m Transition Dipoles g m g x E F m 1 7 397 zu 15 4a m m an Flgure 520 7 rpm n y u mm mm m ujwmimv u mumv m 1nijvrpm nr39mmph v1 Fig 2 5mm dam Mm mm mam mm mmquot kulmmrmndenhpnmrnmlh n m w lht 13mm m 5 mummm A Onenminn a donut m mcepmr mm mm m mva uH F mmm 7 umsilian dipnlu m mumn anal mm m n amnion I is mum L wrymm K P ugmnu Mm res r polmiuuon of namesum upon energy mm m Vurl Lud 1 Lmm Twuw or D xmeptm xqux m m a mu mu 139 w s m typlm mgmg mm indicating a pmmml for massmk 1mm 0 di imm Imngmg chian D 4mm A Mmptm m admin em misilm quotprinted in pamlsian from R21 3m FRET Fluorescence Resonance Energy Transfer Rate kt rate of rxn ifetime of donor k 1 1 R R 6 id t d 0 Rdistance between fluorophores Ro Forster distance JO overlap integral ForSter dlStance K2 transition dipoles of R0K2JAn394Q1697102 fluorophores nrefractive index of medium Qquantum yield lintensity with FRET transfer Efficiency E lointenstiy Without FRET Quenching E 1IIo sadabsorption of accepter with Energy Transfer donor E3ad71 8daquot1 adquot2 a7V2391 sdaabsorption of donor with acceptor I whv gthv Cleavage a NOFRET 5 Enzyme assays mm on FRET prh dpl Fhmlugenlc m are syntha wn n mm dam mm scmpmr mo ecuits are mcheclmmo no a g ammomas samnarvaemmmuesma ccazeo wmuu me FR39ET dislance Upm damage we F Er emaency drops 9 mo a quotmany of donut AncreaSh and ms nmysny a a m Fla mama strains Fr amp rammed humcg nwus mmunmsay for pa ymPLenk mu gen Two munoclunzn un bm es mwsw aqamst m2 antljen and Inte ed respmway mm new and accepxor Mlloropharf FREY 5 observed omy SZOLLus EY AL n me mesa nae or me amlgm winnss humngeneuus K G 7 F WWW N0 HM m W m m mm D Ihe umnbe ed an gen Fluorescence Anisotropy Plane polarized light to exite detect linearly polarized light Any motion that occurs on the time scale of the lifetime ofthe excited state can modulate the polarization Hence this technique is used to measure size shape binding and conformational dynamics IllL39lLICIII light Dulrclm Figure 821 Umquotlimuu iwmn mm in ll1ll quotHUII39N mtu palmnunquot Altlvi Iquot FRET with Anisotropy Al J A onosyuy 13 mo NIIP39I Id lt Fret Apps to Bilayers GM1 toxin GPI anchored proteins GFP Homo versus Hetero Fret a Cuncemraunn depo aHzauon FRET b Convenhuna FRET c SPT Excnauon Ermssmn Excvtahun Enwsswon 39 H Cw O Polanzed emwsswon quot3 r T Wma d nur V H dHue SDWUUH I S eymssmnm dl ule r J 5 J solutlon 7 N Redrshmed 7 wN Depommm by u ancepmremwssmn lt C MKK homoenergylmnsler 4 H7lt d quotquotaccepm39 Y i Con nement 8 L mcnncemrated W K I energy ransferm OS V kzune J 4 50mm concentrated I solution q 1 x r 3nu nm nounn lransfaw I Y W quotI H H Hm mm mm an be FRET uorescence resonance energy transfer 17 A i D A AT runst f ITEKZ 57 hamp RD r01152j3lt0D11394quotG Flunmsrcncc I Wmdcn m m 2 Onsmzum m m mum mtmsula m dcmr m accenv anmnmenl amunmn 1 7 n9 hnunmm mu m 1 u we arms berm m mmquot man m m mum mgunwnwmnumwms HG mn sw ns u munanwehvmwmhslram Mm um Immunn mman m m dmvnr m 1 f FD mmquot IA x2 cos 0L 7 3 Cos B cos 39ylz A DavsonDanielli Model Exierlor i ixf lxii kf lt k i nunnnnnnnnnnnnnnnnnn Lipoid W u 1939 a A 1 Interior 405 9 9 atquot gg 90 v 4339 1 g o 00 705 B Robertson39s Unit Membrane Protein m QQQQQQQQQQQQQW Proiein VJ W I any I xquot on 0N2 an 0quot 1 ifa HWmer HWQWHQ WWM J 3 ANNULAR V PHOSPHOLIPID E J K I 1 N s u gt g w L vUJOkhMTM 4 lt gt JJ w OHMquot 0H ruMuHoHuH 7 gm 0 f RN EZZ E SquotE LANMMR l ANNUIAP HAFr CHOLESTEHDL FHOSPHOLIPED Membrane Rafts Membrane Microdomains Raft is a specific type of microdomain sphingolipidcholesterol rich region Separation of discrete liquidordered and liquiddisordered phase domains occurring with sufficient amounts of cholesterol Microdomain formation is believed to be involved in following cellular processes Cell sorting Signal transduction Endocytosis Calcium homeostasis And others Rafts liquid ordered domain lipids are fluid in that they have a high degree of lateral diffusion but the acyl chains are closed packed and ordered Glycosphingolipids particularly sphingolmyelin and glycosylphosphoinositol GPI anchored proteins preferentially partition into rafts The debate Rafts in model membranes vs Rafts in Biological Membranes Origin TritonX 100 insoluble components isolated from biological membranes Detergent Resistant Membranes DRM Does DRM always equal a raft T ltlt 39I m T NTm T gt Tm ri on can sou iize c o u R3355 Lolo lc gehol 39b39 DOPC h 39 ME till 93M Detergent Micclles l l ozm i aim We 0 0 w i l we 95 020 oo i mm m gt 5 i at H W O More Detergent Missiles z e a at a Mcmcmer m l l l l rim Carr30 03ng till alt all r whammc ullmummn til the cll39ccl vr icmpumtmc mi an h At Ts m m4 w T 1mm Mm lung mine We l l m in nm In er I well c e o I I NW hull mm mm mm M 1m Hp rem men we tcxl H lc H Ind d l 3 men mm mm in lllllg mm IM ll mm cnncvmimmn m i mm Sphingomyelin cholesterol interactions cm Pmquot Lupm a 9 H NHJKAAMAAAA sVv DWW Sphmgomvelm 7 a e HO H am W m i a VAVAVNmVVAV POPC Ao r0 0 l f 65sz g x mlmummhlc Mum Mum win rmly Mnmhrmnu uh mm o gum mummlhr mm m uw ceucc Inltmwopy 4 umm summwmral NMk Wu mmmm m mum mum um and mum Mumu 4 Mumh mm m wwlc mcmh mm v mm M L W Wm we dcumul w Wm NW N a x ml dvcubed m m humming mmpmmmb nl39l w m Mammal m 24 w w a u um chm mm m w w u x Ul hyl C IVIquot39M1IWA Cholesterol Available came a wwwsciencedireacom cquot BuMumnm a 5mm m um 2mm 7 ms mum menmnte mgmu REV BW Mg x hth vhme mingle dwmmg m mm quotmm uwuvhnmcs m lle39 rm mul um um m 25 w mm A39mgm n uuhculc Hun uphum Hm mummy my mm mm mm vhuw m me Hammad mm mm and m lulu phase e A I SM and why mgmxy mucth m Mequ M mum lwml mm m w 1 lt WM 1 December 2005 Available owl nc m www3lcncndlrclcom ELSEVIER leiiimn ex I loplryklci Mm 17 Illllir m liur luwvlwwicmnliminth Prelim Special issue Lipid rafts 4 reviews on domain formation in model membranes and physical properties that underlie raft formation 2 reviews to describe techniques used for studying rafts FRET and uncertainty for detecting rafts in cell membranes Raft Function in Cells 4 on signal transductiongE receptor signaling Growth factors Ras signaling Ceremide Raft function in apoptotic signaling 3 reviews on raft involvement in Endocytosis mammalian viruses bacterial infections bacterial toxins 2 reviews of caveloae Membrane raft Organization DRMs detergent resistant membranes DIGs detergent insoluble glycolipid enriched membranes GEMs glycolipid enriched membranes TlFFs Triton insoluble membranes Raft is more generic as the microdomain L can be caused by protein l l notjust physical properties of the lipids themselves W M ll 4 3 39 Caveats c ages Sphingomyelin ooooua Mia Scoooo u l L S Q caveolin Decode o yOOOCU Cholesterol Snriamlly Speci c kinase pliosphalinid mm mm a A l E c n mmrmr mill ML 1W Xi ll Mil o r T7 drill at algal Caveolin1 caveolin2 caveolin 3 hemagglutinin and GPI anchored proteins serve as markers for raft formation cla l39hrin independent dependent r afT dependem H coa red PIT Cdc42i RhOAl caveolin Epsl5 Dyn l Dyn endosome CGVCOSO e Figure 2 Multiple palhwavs of endncvtosis Different types oi imaginations occur at the plasma membrane to mediate membrane endocytosis Many surlace receptors are internalized into coated plts by a clathrinrdependent pathway Clathnnalndependent raft V V V V V v v but both are A A A and ramm different small GTPases All pathways lead to the endosomal compartment while caveulae fuse with another sorting compartment Limit 1 ultrtlttl lllllllljllllll ll Jlll italic 20M 5 247154 Elaclwell Munisgaard tlii Illl ll Ill l llilillllmlml in Review Lipids as Targeting Signals Lipid Ralts and Intracellular Trafficking Ava m mlvv WMmum m mm mm in 3 A liquid domains enriched in cholesterol and sphingolipids 7 large 50 nm in lmly mm mm M39 M B lipid shells small dynamic regulated processes C mosaic ofdomains maybe regulated by cholesterolbased mec 39sm D small dynamic multimeric lipid assemblies dynamic and transient A O Shell smu Shell Ran meeln motelquot 1 B v v r H v Rm 5 low armIcy mm mm pchowesterow gSpmngumyelm oPhosphohpld GSL Anmgtody em Cytoskmemn GPLanchored Proteins mvolved m mmem swgnal transduction a if 7 WE 100006 I 00 b w w oo o ODMZOOOOMOWJooom 7 L1 mu m m hm mvmumamz mm m m man man p hm b nu ma am 39am m mm m mm m lt um mmm D154 Wm mum in mm mm Um M an cub ummmm mmmm m quot1m mum 4gt 4 Cmske ng a Cmslsnng C H i 39 4 0quot Laws hmmng Preestl ng damam Tlgml undmg Fig 1 y 39 39 m n mun u y m v I v 39mcmhnmc The scanld n c x n m mmw mm pmmmc n curwd canngumion Protein sorting lable 1 Free energy ol melittin transier Hi39IKi melittininrlueerl leakage lor large unilamellar rresirlas h Lipid AG Parrcnlaucoi kmiimnii twinge EPC 7r6t0i mm Ei C rnniastnrnHirl 6r4t02 2 7 03 Spninuornrntinanotmtnro ill 45 tr to tr Ei C Jhnspnatrnyisanno 35 4903 519120 rs 4030 titan 39Ahbrvrratinrrs AG i iraeerrergyai narrate EPC egg phosphatidvlctrolirrs M d irmpnittsmhirr a raraamrarm iron on ran Melittin 26 aa cationic bee venom charmels Role of structural and mechanical properties of bilayers on peptide lip id partitioning Jumpingtoralts gatekeeperroleotbilayerelasticity 316 AGquot kcah mol 2 4 i quota I I 43 8 r r r 500 t 103 i500 2000 Ka rrtrn enr r as meant Farmi Piaoiminiserernitrars eri uliarmcintirrMathsmqrerrra at rrnoctrantrrel protsrn rir 39llrr i iVii39iii rrta irnr rsrds are 39rrne39rnrr ii the ltlll d the trimmer ramrressrhrtny modulus lit nurrrtr irrr arde39ci rrraeasrru varianr to am em unwrarrwrourme rrrcrrra EPCiSlratuiuiEstarrni 1i EPCrhnimmi EM 1 spirrrnrmvsirrerniartmi TM hinting at NPR In it springnrnyaiirr Miami is tan small in daarrnirre 15 warship 3 l39ta rrrtrrntrerr 2rd tamer time s 5 an in biii tttir i in in amstrltrumt iiFi39idHa respsanelyr itaeratstenimrn iii B ir 11 1 mixture of DOPCSPMCHOL the detergent insoluble fraction has a thickness that is 9A greater than that of the DSM Role of Bilayer thickness in proteinlipid interactions possible role in sorting of proteins via hydrophobic mismatch of the transmembrane domain TMD Hydrophobic mismatch ifthere is a mismatch between the length of the TMD and the hydrocarbon thickness then the bilayer would need to deform to prevent exposure of the hydrophobic amino acids to water This would be energetically unfavorable So if the protein can move to a raft of different thickness there would be a driving force for such partitioning hllsysrslllllsdsqumsl miman n1 rhrlriwul m m n my mmai M nawi 507 mm mum Wm WM mini m rw lESl mimimmmnimr Bio significance in GOLGI proteins with short TlVle reside in non ra regions whereas proteins with longer TMDs reside in ra ions destined to the plasma membrane rich in cholesterol and SPM Length of TMD has been indicated to be an important factor in controlling protein trafficing Experimental studies of peptide sorting by length 015 I P723 1 e E Fze E a DSM bl DRM a 0107 E i E I l s E 0057 pg 0 39 A 329 48 A E W TES res Figure 3 illustration 0 peptides with different lengths in detergentsoluble mem branes lDSMs and detergentrresistem membranes lDHMsl Peptides N3 Flguve 4 Ram or ecuJ c to oml llpid n dotcrgcrl39 Sclumc membranes iDsMs KKGlLAlAWlLAlAKKA and P29 KKGlLAlsLWlLNgLKKA are shown in bilayers and dctclgcm es manl membranes lD Msl tor bcin P 23 me P 29 at 4 C and with widths corresponding to DSMs la and DRMs lb respectively that were isor 3quot C Periwinklequotquot0th 165 lated from l l 39 39 39 l r 39 39 biiayers 65 The peptides were designed so that the transbiiayer region 17 and Table 2 Mulefraction panitiun coe icients and apparent hee 23 nonrpolar amino acid residues or P723 and P729 respectiveiv matched the energies c transferforpep des and npids from DSMS m DRMS hydrophobic thicknesses oi DSMs and DHMs respectively obtained lrom Xray at 37 acet diffraction analysis 42 Phosphollpids are shown as circular headgrotlps dark blue sphingomyelin light blue DOPC attached to wavy hvdrccarbon chains Molecule Ku AG ltcalmol 1 39 39 I l P 23 028 t006 082 013 each peptide the open central box corresponds tn the transbllaver uhellcal core P49 153 t A 030 t 016 region and the red boxes correspond to the hydrophilic regions which each cons mmiesmml 727 I 071 tain two lysine residues that anchor the hydrophilic ends at the peptide to the DOFC ms t 003 122 i 039 interfacial region Similar peptides were used in Ref 54 except that each hydro Sphmgomye in 425 1122 037 1 mo phllic anchoring region contained two tryptophan residues and the transbilayer segments contained i7 21 or 25 amino acid residues choline Kg partit on cocltrcicni F 23 transoilaver peptide With 23 amino ends 9725 ransbllaver peptide Wlm 25 amino acids Data areaken rrom Rel 65 Thermal kT 06 kcalmol at 37 C Big Question We can see rafts in Model Membranes GUVs or Supported Lipid Bilayers LM but how to study in cells Do rafts really exist in cells Are they static large structures Are they small transient structures FRET and FRET based Microscopy Techniques 4 n z FRET uorescence resonance 1 mmquot k K I V E 3 energy transfer E kr T T q kn R0 mum gnlr l W E k kT kr kn s he lrnmmau mmmm39 my Mum am uan 5 mm W H 2 mm H quotMaths 4 huvnnmnbmmv lhHnnaman mamaquot 1 mm Hm lmmmnn movth my he mum mm m Wvumm u n WM w 1 Us 9 wh b m u n m mnemer K mm 3 3905 Buys39yl f FDW HM Amwpnun A 1 Flunrurrnrc nuan I mrlcnglh A gt11v FRET 391 m P hv Cleavage who I B 3 NOFRET Ills T m 5 mm 3 mm mm m mm A 5mm a my 4 m m m mm Nam m I WWWWMV m and mung quotWWW o 0 um um m FREI Mm u m mm m rm min m In new m Mava M ml Wm M m WW 3 Ab Antigen Ab WM N J m e FRELDrm vanrgeueom muwmsuy39nr pa ynaplem aw gen mummmm ammu un mm Wu me Hugm and labeled V e ywmxdmnlmmwczemuHum my FREY saL wwdm y mug r AL m m pmenrequzm U M a rm mm amnm mm h wdaad WW m m wry w 1 o rum a 3 m Q Um Q gra wa iu mmm J mum iul Nv 4 mM xx 6X6 gx k b Convennona FRET EXCIt HOH EHHSgtIUH Excnauon a Concentration leperanzanon FRET C SPT vawun MV O Polanzedenusswon lt7 f NonnaMonor B nd ulesomuan B elmssmnm dnute A suluuun 7 14 Redsm ed i x Depa anzawmy acceptor emwsswon i 7 lt11 homnenergwransfer lt7 V39adunor39accepmr A Con nement 3 m concentrated energy m39wm39 W W5 xzon so on C 5 quot DMIDH e um l om men H W 1 I 1 1 l 7 l h o 300an FIGURE y a mreed wmemmna Y 1 4 0 Rn Huxan FRET wmm an we Effects of Cholesterol on Membranes Physical Properties Removes gel to liquid crystal phase transition New intermediate phase called llqi39d ordered ordering of the membrane lipids due to condensation closer packing less transgauche isomerizations Cholesterol thickens bilayers with 16 C or less Cholesterol thins bilayers with 18 C or more H0 Choleswmi 1g 55 SpaceMlng models huwmn the numl Ilkzly pmilinn I clix vkhxcrol in a mem hmnu rclanvc to he phosplmlipids Modulation of physical properties and minimizing permeability to small molecules 6742 Biochemistry Vol 3 No 29 1992 Suppression of phase transition to LC state Temperature 39c u o a o E IO 20 t Mom Choiesterci noun 1 DMPCcholsslewl phase diagram Filled poms cor rupond io discontinuities in experiments using a 5PC spin labs o and a l PC biradical label 7 Sankaram a Thompson 1991 o am am in 39 Figure 4y The lines drawn thmugh 111 points represent our best judgement NMR EPR uorescence DSC Endoihemi m in 10 u a m in TnllkrllumquotC nLuxE z DSCheumg icaisumummmuriiiiiimmi will nMK mm in mm immmn mm kzdmwifmm imii FRAP Flourescence Recovery After Photobleaching Used to determine Viscosity relevant to effects of cholesterol lateral phase separation and RAFTS bleaching redistribution Eiochemistry 1991 31 6739 6747 Lateral Diffusion in the Liquid Phases of Dimyristoylphosphatidylcholine Cholesterol Lipid Bilayers A Free Volume Arialysiis39r Paulo F F Almeida Winehil L C Vazll and T E Thompson Deparlmem of Biochemmry University of Virglnla Charlottesvllle Virginia 22908 and MaxPIanckImrllut lr biophyrlkalisch Chemie Ponfach 2841 DWidDO Gbmngen FRG Received March 23 1992 Aasmcr The technique of fluorescence recovery after photobleaching is used to perform an extensive studycfthelateraldiffu 39 39 39 39 39 39 t p nlmoml 39 r smelling r L gt u L t r J L Macedo t vi 1 k quot39 1 39 In the liq d quot quoti39 39 quot regiuuthe temperature dependence of the diffusion coef cient is in excellent agreement with current theories of generalized diffusivities in e k A I A m r J r r Iquot the idea quot L quot 39 N we aim i to occupy free volume On this basis a general interpretation of the phase behavior of this mixture is also proposed F0 2 a Fm C l E g I m u C l m U I 3 5 FED 2 gt time 1 NBDrphosphahdyleiIanulamme n x M NEDPE i ll Hc i inrowcmcuun v N v I 1 CH 0 quot 0 D CH f i We 7 JiFiL l Ci LCHILCHr 1 CH O K Wlx tcgcp Permeability Hypothesis gaucheTrans isomerizations lead to small gaps or holes of free Volume space The idea is that the constant movement of the bilayer Will make a series of connected holes to allow small molecules smaller than glucose to pass Cholesterol reduces the number of gauchetrans isomerizations by ordering the hydrocarbons thus limiting the permeability M In In An 0 thnlrsrrrnl 11 57 l39mthunul volume n a lunntc 1 lequ r m x x u L we palnulnnamalndunn39l PC mm m I AYC 30 mole c humeral m PAPC A rhodopdn immml V on I rhodupwin PCMhadops mum A 30 H mm rakequot mm M Slmumt mm a J Lamquot RwuArnmrw m um n gt1 lumen manWm 27 uyxm 7723 mum pnwmaa by M 31 l mrm Q 1 gt 1 mom chnlnvelnl M Slraumc mum and Increased elastic stiffness allows membranes to get bigger and withstand stress ie erythrocyte membrane 4550 cholesterol 39ncreased viscosity changes the diffusion rate of membrane proteins can also effect function of membrane proteins NaKATPase and growth rhodospsin etc Cholesterol Movement Between Membranes collision mediated partitioning although cholesterol has low solubility in water sub nM it is believed that cholesterol will partition in and out of the membrane like a detergent or fatty acid and can transfer between membranes via this mechanism Other interesting facts cholesterol rotates along it s long aXis with a correlation time lOOps phospholipids rotate with longer correlation times lOns Therefor there are no long lived complexes between phospholipids and sterol Cholesterol Location In Cells Plasma membrane 3545 mol ER 1012 inner mitochondrial lt 10 Cholesterol is also heterogeneously distributed Within given membranesorganelles Question becomes What is controlling separation and trafficing of cholesterol Physical properties of the membrane 2 03V 1 I I I Cholesterol prefers E 5 membranes of PC over PE 5 395 020 PC hydrophobic effect as g l discussed early for PE E 3 010 39 bila ers 53 E PE y C 5 O i l I 3 10 20 3O 40 53 Time h Fig 52 li lCnbulinn ol39 smztll soniutitetl PC rexiclcs containing cholesterol with huge unilumcllur PE ics clc containing no ChUlBSlCIDIi A a function of urns the vesicle popular ions were sen Alekl and the Ch0l ErnliphOSphulipill molc rzttio MIN determined It is evident that Chulextcml prefers to stay In the P vexich 2 030 U 45 gag 020 5 0 a gmo 2 O c u 01 3 10 20 3o Timeh Fig 53 Incubation as in Fig 52 but in the presence 0 M guanidinum hydrochloride GuHClL In he presence of he chaotmpic agenx cholesterol distributes readily belween 40 the PC and the PE membranes with no evidence for preference enauphos Sucmse Fig 56 Rzlanansmp buchenmechulummlcommnlmnnal mumumcgnmga and am we Thl disks me EtnaMu scam1quot m chulzqgml mum on g mum mainquot n cr mmunum wllh diguunin The mg F J wmcm 112er um J Hm men xl uv ansthlnImth T Hmcsgtcynnd LD Inuzu9almxLH Ish mm min mu15 I 1 compm wlmchulcucml K H5 P mam 50 0 0 0 m mm cwm NI 5 m SCS quot mocmmu Mm m Nucuus I 4 9 Am swan Tawnme Hzmzmmm awn co Hzmznmm mmzz Common Sterol Structure 2E 27 25 a 14 WA Numbenng ohm chulasleml 1an system wolnstane MN BaChMGSWVIa snucuue and chaillorm Clulmlm m clmlesleroi H10 H30 H W Coproslam or 5 ancdusxana Struolum and damn 00039 OH on Challlorm n1 cnmale HO Cholesterol l 18 steps biologically HO Lanostuml H0 Ergosxerol Question for exam why doesn t lanosterol or epicholesterol have the same effect on the structural properties of phospholipid bilayers Cholesterol Modulates Membrane Structure dynamics and or function through three major molecular mechanisms Membrane protein function Via sterolprotein interactions internal properties of the bilayer and cell membrane hence can also affect protein function alters lateral distribution of components in cell membranes raft formation Mammalian cells require cholesterol for growth mycoplasma mycoides also require cholesterol 05 cerevisiae yeast require ergosterol Cannot substitute cholesterol for ergosterol in various cells therefor there must be some speci c proteinsterol interaction that can discriminate Studies on NaKATPase resp0nsible for maintaining the Na and K gradients single greatest user of ATP in many cells Found in plasma membrane hightened activity with higher concentrations of cholesterol Extrapolation to zero cholesterol from experiments indicates enzyme would be inactive if the plasma membrane had no cholesterol Prawn thsphnrylannn m omnmmm dung am no Nu m K am my K Kn Ike Dephnsphnrylmnn and k m me Ike Ce K m Tnp is m emu membrane mam man membrane mm nomcm httpwwwvivocolostateeduhbooksmoleculessodiumjumphtml Other proteins affected by cholesterol NaCa2 exchange ATPADP exchange glutamate transport GABA transport mediated lactate transport acetylcholine receptor galanine binding to galanine receptor Speci c binding to protein binding to protein in the bilayer annulus of sterol omodi cation of bilayer properties Ergosterol 505 1 45 39d quot0 do 399 a d 8 40 i g i i i i to gt 5 35 50 m solo quotB m l I t t lt i I i 30 IIl i I l I I l p 25 j 39 I I l D 10 20 30 ergosterol conc mol FIGURE 10 Partial phase diagram of the DPPCJ crgtrsterti membrane Midpoint of the transition from AMT curves Fig 5 l onset or end 01 transition in MIIT curves l 1 obtained by inspection olithe depaked spectra versus ergosterol concentration Fig 8 0 end of transition in DSC data shifted by TC 0 obtained by inspection of the depaked spectra versus temperature F 7 l A 1 obtained from lIltergostcroll curves Fig 6 CW obtained From spectral subtraction I the onset oftransition in MltTlcu cs For MLDs having ergosterol concentrations of 25 2735 or 30 mol u it Einphyslcaldoumal Volume 85 Marchznns 17994503 1789 The Effect ofquot a 39on quot39 39 39 Bilayers A Deuterium NMR and Calorimetric Study Phase Behavior OI Lipids and Slemls I761 PROMOTERS INHIBITORS o 1 V i i Andms eno one new J fgt 5 Cholesterogo x 39 Dmydrcmolesterol o H 1 0H FIGURE 1 5mm uuuum dmwn Iu highr P gt Chol m NJ A k v e quot9 light asxignmanl of hydro n hydmxy and HO a V V J 7 39mm muhyl wroup m euher m a a 77mm m or 3 w prrm mm 9139 Lb LcnvI Dimmmormf ring Ho 4 Nx Epmuxeaewx due to duuble bonds is no shown for mu r01 4 L39 Wig Somme ergommlmd Imusk ml KI iquot z 25Hydmxycholesterol a f Ow ma copmslam OH
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