pdb101.rcsb.org

LessonPlanCarryingCargo:Exploringnon-covalentinteractionsbetweenproteinsandsmallmoleculesinbloodtransportGradelevel:9-12Objectives:Aftercompletion,studentswillunderstandhowthehydrophobicandhydrophilicinteractionsbetweenproteinsandsmallmoleculesareutilizedintransportingoflipidsandothersubstancesinthebloodSummary:StudentsusethedataandtheinformationfromtheRCSB.organdPDB101.RCSB.orgwebsitestolearnaboutthefunctionofserumalbuminandtoexaminetheproteinin3D.Theyexplorethestructuralfeaturesandthenon-covalentinteractionsthatareemployedbytheproteintocarryfattyacidsintheblood.NGSSStandardsScience&EngineeringPractices:– Engaginginargumentfromevidence– Obtaining,evaluatingandcommunicatinginformation– Developingandusingmodels

DisciplinaryCoreIdeas:– LS1A:StructureandFunction

CrosscuttingConcepts:– Patterns

Inaddition:Studentsgainexperienceincollectinginformationfromadatadrivenwebsite.Theypracticeanalyzingandcollectinginformationthroughapplyingvariousvisualizationtechniquestothesamesetofdata.Theyexploremodelsgeneratedbyscientificexperimentsandusetoolsdesignedforadvancedscientificanalysis.Materials/resources:• ComputerwithInternetaccess.Resourcestobeaccessedthroughoutthelesson

• MoleculeoftheMonthonSerumAlbumin:fromthetopmenuofpdb101.rcsb.org,chooseMoleculeoftheMonthandaccessSerumAlbuminarticlebytitleDirectaccesslink:https://pdb101.rcsb.org/motm/37

• SerumAlbuminStructure1e7i:gotorcsb.organdenter1e7iintothesearchbarDirectaccesslink:https://www.rcsb.org/structure/1E7I

• Mol*tutorial:fromthetopmenuofpdb101.rcsb.orgchooseBrowse,gotoStructureandStructureDeterminationthentoVisualizingMoleculesandlocateamongtheLearningResources:ExploringPDBstructuresin3DwithMol*(MolStar)Directaccesslink:http://pdb101.rcsb.org/learn/videos/exploring-pdb-structures-in-3d-with-molstar

•Studentworksheet(providedwiththelessonplan)•Possibleuseofprojected/sharedcomputerscreen

pdb101.rcsb.org

Preexistingknowledge:Studentsunderstand:• proteinsarepolymersmadeupofaminoacidsandthatthepolymerchainformstheelementsofsecondarystructureandfoldsintoaspecific3Dshape

• aminoacidssidechainsfallintothreedistinctivegroups(hydrophobic,hydrophilic,charged)dependingonhowtheyinteractwithwater,andothersidechainsandmolecules

• basicsofthehydrophilicandhydrophobicinteractionsandtheconceptofcovalentandnon-covalentinteraction(thisinformationmightbealsoreviewedbeforethefirststepoftheactivity)

• themainconventionsin3Drepresentationsofproteins(cartoon,surface)andatoms(ballandstick,spacefill)

Note:Thisinformationmightbetaught/reviewedusingPDB-101videoWhatisaProtein?(pdb101.rcsb.org>Learn>Videosorhttp://pdb101.rcsb.org/learn/videos/what-is-a-protein-video)Inaddition,TedEdlessoncreatedbyAlisonAckroydhttps://ed.ted.com/on/xdk2WHce#watchoffersquestionsforassessment.Preparation:

StudentsfamiliarizethemselveswiththecontentsofthetutorialExploringPDBstructuresin3DwithMol*toallowforfullfocusontheactivityduringclasstime.Thiscanbeassignedashomework.Thetutorialshouldbeusedasaidthroughoutthelessonaswell.

LESSONACTIVITIES:Studentscanentertheiranswerandevaluations/predictionsontheenclosedStudentWorksheet.Studentscanworkindividuallyoringroups.Theteacherhelpsstudentsalongandmakessuretheystayontrack.TheteacheralsofacilitatesinclassdiscussionsandassessmentactivitiesCOLLECTINGINFORMATIONIntroducingtheFUNCTIONofserumalbuminwithMoleculeoftheMontharticleStudentsaccesstheMoleculeoftheMontharticleSerumAlbuminonPDB-101.TheyreadonlytheIntroductionandCarryingFattyAcidssectionsandanswerquestion1(asnumberedonstudentworksheet):

• Whatisthefunctionofserumalbumin?Whyisitimportanttohaveappropriatelevelsofthisproteininthebloodplasma?

Answer:

• Serumalbuministhecarrieroffattyacidsintheblood.Fattyacidsarethebuildingblocksforlipids.Lipidsformthelipidbilayermembranesaroundandinsidethecells.FattyacidsmayalsobebrokendowninsidecellstoformATP,animportantsourceofenergyforourbodies.Whenweneedenergyorbuildingmaterialsforbilayermembranes,fatcellsreleasefattyacidsintotheblood.Serumalbuminpicksthemupanddeliversthemtodistantpartsofthebody.

pdb101.rcsb.org

UsingtheRCSB.orgStructureSummarypagetocollectinformationabouttheSTRUCTUREofserumalbuminInthispartofthelesson,studentswillexplorea3DmodelfromtheProteinDataBank(PDB).IfstudentsarenotfamiliarwiththeProteinDataBank,theteachermayintroduceitbrieflytouchingonthefollowingpoints:

• PDBisadatabasecollectinginformationaboutthe3Dcoordinatesofatomsmakingupproteinstructures

• ThestructuresaredeterminedallovertheworldbydifferentscientificexperimentsthensubmittedtothePDBarchive

• Thereare4PDBdatacentersallovertheworldthatcuratethedata,maintainthearchive,andprovideaccesstothemalongwithtoolsforsearchandanalysisfreeofcharge.OneofthecentersisRCSBprovidingaccessthroughrcsb.org

• Rcsb.orgisahigh-levelresearchresourcesostudentsshouldnotfeeldiscouragedifinformationishardtolocate

• PDB-101istheeducationalportalofRCSBthatcreateseducationalresourcesbuildingonthedatafromtheProteinDataBank

StudentsaccesstheStructureSummarypagefortheserumalbuminstructure1e7i.StudentsusetheMacromoleculessectiontoanswerquestion2:

• Howmanychainsdoestheproteinhave?

Answer:

• Theproteinhas1chain(chainA)IntroducingStearicAcidStudentsgototheSmallMoleculessectionontheStructureSummarypage,analyzetheinformation,andanswerquestion3:

• Howmanytypesofsmallmolecules(ligands)interactwiththepolymer?Answer:• Onetype,stearicacid

StudentsaccesstheLigandsummarypagethroughtheligandIDlink,analyzetheinformation,andanswerquestion4:

• Whatatoms/howmanyofthemistheligandmadeof?Howaretheyarranged?Answer:• Theligandismadeupof2oxygenatomsand18carbonatoms.Theoxygenatomsarecovalentlyboundto

oneterminalcarbonatom,andthecarbonatomsarearrangedinalongchain.

Studentsreviewtheanswersinclasswithteacher’sfacilitationandpointtosupportingdataEVALUATINGINFORMATIONANDCREATINGPREDICTIONSStudentsevaluatethesmallmoleculemodelstructureandpredictthekeyfeaturesofitscomponentsandanswerquestion5:

• Ifyouweretousethewords“head”and“tail”todescribetheligand,howwouldyoudescribethearrangementoftheatomswithinthem?ThesmallmoleculeiscalledstearicACID.Whichpartmakesitan

pdb101.rcsb.org

ACID?Howwouldtheacidbehaveinwatersolution?Whatchargewoulditcarry?Howwouldthetailbehaveinthewater?Woulditcarryanycharge?

Answer

• The2oxygenscovalentlyboundtothecarbonatomscouldbedescribedasthe‘head’,thecarbonatomsarrangedinalongchainmightbedescribedasthe‘tail’.TheACIDpartistheCOOhead.Itwillbeionizedandcarrynegativechargeandinteractstronglywithwater.Thetailwouldnotcarryanycharge.Thetailwouldbenotinteractingwithwaterandwouldtrytofindotherhydrophobicparticlestoshielditselffromwater.

Basedonthefirsthypothesis,studentspredictthetransportenvironmentfortheligandandanswerquestion6.Theteachermightdisplaytheimagebelowontheoverheadprojectorforreference.Theteacherexplainsthatthestudentswillcreateaschematicmodeloftheinteractionstheypredict.Themodelcanbehand-drawnorcreatedusinganycomputersoftware.

• Whydoesthislipidneedatransportproteintobecarriedinthewater-basedbloodplasma?Whattypeofaminoacids(hydrophobic,hydrophilic,orcharged)wouldneedtobepresentontheoutsideoftheprotein?Whataboutinthecoreoftheprotein?Whattypesofaminoacidsidechainsinthecarrierproteinwouldthe‘head’interactwith:polar,charged,orhydrophobic?Whattypesofaminoacidsidechainsinthecarrierproteinwouldthe‘tail’interactwith:polar,charged,orhydrophobic?Explainwhy.Drawamodelofatransportproteinwithstearicacidandhighlighttheseinteractions.

Answer

• Thehydrophobictailwillhavetobeshieldedawayfromthewater,that’swhythismoleculeneedsacarrierprotein.Theproteinwouldneedmostlypolarorchargedresiduesontheoutsidetointeractwithsurroundingwatermoleculesandhydrophobicresiduesontheinsidetoaccommodatethetails.Theoxygensintheheadcanbeexposedtothesurroundingwaterinthebloodplasma,creatingH-Ohydrogenbonds,andcanalsocreatehydrogenbondswiththehydrogensonthepolarandchargedresidues.Examplehowamodeloftheseinteractioncanbedrawnisshownbelow.However,studentsshouldusetheircreativityandoriginalideastodevelopthemodel,soshowingthemanexampleisnotrecommended.

pdb101.rcsb.org

ENGAGINGINARGUMENTFROMEVIDENCE:SharinganddiscussingpredictionsStudentssharetheirpredictionsandmodelsinclasswhiletheteacherfacilitatesthediscussionandasksadditionalquestions.TheteachercanshareaGooglepresentationwiththestudentsforthemtoinserttheirimages(ifcreateddigitally)orphotosoftheimagesifhand-drawntosharewiththeclassviaoverheadprojector.Theimagescanbeusedinsupportoftheirarguments.Atthisstagestudentsshareargumentsfortheirpredictions,buttheydon’tknowwhethertheirpredictionsarerightorwrong.COLLECTINGMOREDETAILEDINFORMATIONANDEVALUATINGPREDICTIONSExploringthe3DstructureofserumalbuminwithMol*Studentsaccessthe3DviewofserumalbuminfromtheStructureSummarypage.Theyexplorethefeaturesofthe3Dstructureandanswerthequestions7and8.StudentsmightsavetheimagesfromMol*touseinthenextstep.

• Whatelementsofsecondarystructurearepredominant?Whatdoestheoverallshapeoftheproteinresemble?

• Evaluateyourpredictionsfrompreviouspartofthelesson:whattypesofaminoacidsarepresentontheoutsideoftheprotein?Whatkindofaminoacidsmakeupthecoreoftheprotein?Why?Youcanchangethepolymerrepresentationtospacefillandthecolorthemetohydrophobicitytoevaluateyourpredictions(seeMol*tutorialsection7and10)

Answers:

• Alphahelicesarethepredominantelementofthe3Dstructure,theoverall3Dshaperesemblesaheart

• Theaminoacidsontheoutsidearemostlyhydrophilictointeractwiththesurroundingwaterinthebloodplasma.Theaminoacidsinthecorearepredominantlyhydrophobictoholdthe3Dstructurestableandtocreatetheenvironmentforthehydrophobictailsofthelipidstobetransported.Exampleimagebelowshowstheproteinwiththehydrophobicitycolorthemeapplied.Hydrophobicresiduesareinshadesofgreentolightyellow,theresiduesthataremorelikelytointeractwithwaterareshownindifferentshadesfromdarkyellowtored.

pdb101.rcsb.org

Assessment:Studentsreviewtheanswersinclasswithteacher’sfacilitationandpointtosupportingdatausingsavedimagesasevidenceExploringthepolymerligandinteractionin3DAsthestudentsexploretheproteinin3D,theyanswerthequestions9,10,and11.Studentsmightsaveimagestouseinthenexttosharewiththeclasstosupporttheirargumentsintheassessmentstepthatfollows

• Howmanyligandscanyouspotinteractingwiththeprotein?(youmightchangetheligandrepresentationtospacefillandapplydifferentcolorstothemusingsectionsinordertohelpyouspotallofthem).Twoligandsareoverlapping.Doyouthinkbothofthoseorientationscanhappenatthesametime?Howmanyligandstotalcanthisproteintransport?Clickonanyoftheligandstozoominandobserveinteractions.

• Whattypesofaminoacidsdoesthe‘head’interactwith?Whatnon-covalentinteractionsdoyouobserve?Whattypesofaminoacidsidechainsdoesthe‘tail’interactwith?Whatnon-covalentinteractionsdoyouobserve?Clickonotherligandsandobservetheinteractions.Whatdoyounotice?

• Changetherepresentationforthepolymertogaussiansurfaceandtherepresentationofligandtospacefill(seeMol*tutorialsection7).Whatoverallstructuralfeaturesdoyouobservewhenlookingattheproteininthisrepresentation?

Answers:

• Thereare8ligandsshowninsidethemodel.Theoverlappingligandshavetheheadsontheoppositesidewhichmeanstheycanentertheproteinineitherorientation.Themodelcaneffectivelyinteractwith7ligands.Note:withthisquestiontheteachercouldprovideabitofbackgroundifstudentsareconfused:Crystallographycapturessnapshotsofmolecules,andsometimes,differentmoleculesinthecrystallatticecanbeboundindifferentorientations.Theoverlappedligandisanexample--insomealbuminmoleculesinthecrystallatticeitbindsoneway,inotheralbuminmoleculesitbindstheoppositeway.Thecrystallographersincludebothorientationsinthecoordinateset.Theimagebelowshowstheserumalbumincrystallatticeandcanbeusedasvisualaidintheclassroom

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• Theoxygensinthe“head”formhydrogenbondswithneighboringpolarandchargedaminoacidsidechainsandwaters.Thecarbonsinthetailformhydrophobicinteractionswithhydrophobicsidechainscarbons.Thenumberofinteractionsandthespecificaminoacidsaredifferentforeachligand,althoughtheheadalwaysinteractswithpolarandchargedamioacidsandthetailsalwaysinteractswithhydrophobicaminoacids.

• Theligandsfitverytightlywiththeprotein(asseenintheexampleofanimagethatcanbesavedusing

suggestedmolecularrepresentation).Possibleimagesavedforthisstagebelow.

Studentsreviewtheanswersinclasswithteacher’sfacilitationandpointtosupportingdata.FINALCONCLUSIONSStudentsanswerquestion12,inclassdiscussionfollows

• Summarizehowtheshapeofserumalbuminisoptimalforitsfunctionoftransportingoffattyacidsinthebloodplasma.Comparewithyourhypothesis.

Answer:

pdb101.rcsb.org

• Theproteinhashydrophilicaminoacidsontheoutsideandisfairlysmallwhichhelpsittointeractwithwaterintheblood.Thehydrophobicaminoacidscreatetunnelsthataccommodatethehydrophobictailsoftheligand.Thefactthatitcantransportupto7ligandsmakesitveryefficient.

REVIEWANDEXPANDONTHEKNOWLEDGEStudentsfinishreadingtheMoleculeoftheMontharticle.Muchoftheinformationinthearticleprovidesisareviewoftheobservationsmadewhileinteractingwiththe3Dmodel.Studentsthenanswerquestion13whichhighlightsthenewinformationtheyobtained

• Whatothersubstancecanserumalbumincarryintheblood?WhatotherbloodtransportproteinsdoestheMoleculeoftheMontharticlementionandwhatligandsdotheytransport?UsethePDBIDstoaccesstheseproteinsonrcsb.organdobservetheinteractionswiththeligandsin3D.

Answer• Theproteincanalsocarryibuprofenandarachidonicacid.Otherexamplesoftransportproteinsare

transferrincarryingironandtransthyretinwhichcarriesthyroidhormone.

Optional:Studentscanexploreadditionalserumalbuminstructuresinteractingwithotherligands(e.g.PDBID6u4x–withibuprofen,orPDBID6mdq-withtestosterone)andexplorethe3Dinteractions.