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Methods in Molecular Biology 2039 Jennifer J. McManus Editor Protein Self-Assembly Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Protein Self-Assembly Methods and Protocols Edited by Jennifer J. McManus Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland Editor JenniferJ.McManus DepartmentofChemistry MaynoothUniversity Maynooth,Co.Kildare,Ireland ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-9677-3 ISBN978-1-4939-9678-0 (eBook) https://doi.org/10.1007/978-1-4939-9678-0 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2019 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:233SpringStreet,NewYork,NY10013,U.S.A. Preface Protein self-assembly describes many different pathways leading to a range of condensed states of proteins that include concentrated protein droplets, reversible and irreversible amorphous aggregates, fibrils, viral capsids, protein nanocages, and crystals. These condensed states are important in understanding fundamental features of biology and severalindustrialprocesses.Proteincondensationisassociatedwithmanydiseases,including sickle-cellanemia,cataractdisease,andseveralamyloid-associateddiseasesincludingAlzhei- mer’sdiseaseandParkinson’sdisease.Theobservationsofproteinde-mixinginmammalian cells leading to the formation of transient non-membrane-bound organelles are revealing significantnewinsightsincellbiology,RNAprocessing,andpossiblyeventheoriginsoflife. Drug development relies on the availability of high-resolution protein structures, and the vastmajorityofproteinstructuresaredeterminedfromX-raydiffractionofproteincrystals. Manyindustrialprocessesalsorelyonafundamentalunderstandingofproteinself-assembly. The production of biopharmaceuticals, food, cosmetics, and even some electronics all involveproteinself-assembly. Understanding protein self-assembly is incredibly difficult. If or how protein self- assembly occurs depends on a wide range of factors, many relate to the characteristics of theproteinitselfandothersrelatetotheexternalenvironment.Proteinscanself-assemblein foldedor unfoldedstates byseveraldifferentmechanismsand ondifferenttimescales, and these assemblies often exist in non-equilibrium states. The assembled forms of the protein can also be challenging to characterize, due to the wide range of sizes over which they form—from several nanometers to tens of microns—and this creates an analytical burden. Forthesereasons,itisoftennecessarytoemployseveraldifferenttechniquesandapproaches to measure the assembly process. In this volume, experimental and computational approaches to measure the most widely studied protein assemblies, including condensed liquidphases,aggregates,andcrystals,aredescribed. Understanding the protein-protein interactions that direct self-assembly is far from straightforward.Themostbasicapproachistoviewproteinsassmallparticlesthathavean interaction energy, which has both attractive (van der Waals forces, hydrophobic effect, dipole-dipole) and repulsive (electrostatic repulsion, hydration) contributions that arise from the amino acids on the protein surface. The net interaction potential is the sum of these contributions. If it is averaged over the surface of the protein, then the effective potential resembles that of a small colloidal particle, and we can use what we know about colloidalsciencetounderstandproteinassembly.Thisviewandapproachworksreasonably well for some proteins, particularly for small globular proteins at low concentrations, and canpredictsomegeneralfeaturesofproteinself-assembly.However,formanyproteins,this simplifiedmodelfailstodescribetheprotein-proteininteractions,particularlywhenprotein concentration is increased or there are small modifications to the protein surface. It has becomeclear thatprotein-proteininteractionsaredirectionalinnature,andthisanisotropy in the interaction potential is key to explain protein assembly. In Part I of this volume, the techniques to measure protein-protein interactions and equilibrium protein phases are describedforbothdiluteandconcentratedproteins. Proteinaggregationisperhapsthemostwidelystudiedself-assemblypathway.However, aggregation is a very generic term that describes a range of pathways, including v vi Preface self-association, cluster formation, fibrillation, amorphous aggregate formation, gelation (sometimes),andprecipitation.Thereareawiderangeoftechniquesusedtomeasureboth thekineticsofaggregationandtheassembledstateonceformed,andinPartII,severalare described. In general, a combination of techniques that rely on different analysis methods areusedtomeasureproteinaggregation.Protocolsdescribinganalyticalultracentrifugation, electrophoresis, chromatography, calorimetry, light scattering, imaging, fluorescence spec- troscopy,andNMRareincludedhere.Thiscomprehensivesetofprotocolsallowtheanalysis of assembled states ranging in size from dimer and small oligomers to large amorphous aggregatesacrossarangeofproteinconcentrations. Amajorgoalinthefieldistodevelopmodelsthatwillallowprotein-proteininteractions and protein assembly pathways to be predicted ab initio. While predictability is currently difficult, several computational approaches to understand protein self-assembly are providing valuable insights and are described in Part III. For peptides and very small proteins, all-atom simulations with explicit solvent are possible if some structural informa- tion is already available. For larger proteins, or for simulations that require several protein molecules, computational resources are still not sufficiently powerful to perform all-atom simulations, and some coarse-graining is required. Some of the most successful models to date are those based on colloids that include anisotropic interaction potentials or “patchi- ness.”Thedetailsofhowthesepatchesaremodelledvary,withsometuninginteractionsto match experimental data, while others incorporate molecular-level details from crystallo- graphic data to precisely describe the protein-protein interactions. Using these coarse- grained models in combination with simulations, experimental data can be described and explained. As these models become more sophisticated, and computational resources increase,evengreaterinsightswillbepossible. Much progress has been made in understanding protein self-assembly, but obstacles remain. Detailed knowledge about all of the phases and states of proteins exists for only a relativelysmallnumberofproteins,i.e.,thosethatareavailableinsufficientpurityandscale toallowexperimentstobeperformed.Asmoreexperimentsonagreaternumberofproteins are performed, and computational tools become faster and more sophisticated, further insightsandpossiblyevencontroloverproteinself-assemblywillemerge. Maynooth,Co.Kildare,Ireland JenniferJ.McManus Contents Preface ..................................................................... v Contributors................................................................. ix PART I MEASURING PROTEIN-PROTEIN INTERACTIONS AND PROTEIN PHASE DIAGRAMS 1 MeasuringNonspecificProtein–ProteinInteractionsby DynamicLightScattering................................................ 3 DanielCorbett,JordanW.Bye,andRobinA.Curtis 2 LightScatteringtoQuantifyProtein–ProteinInteractionsat HighProteinConcentrations............................................. 23 MahletA.Woldeyes,CesarCalero-Rubio,EricM.Furst, andChristopherJ.Roberts 3 QuantitativeEvaluationofProteinSolubilityinAqueousSolutions byPEG-InducedLiquid–LiquidPhaseSeparation .......................... 39 YingWangandRamilF.Latypov 4 MeasuringProteinSolubility ............................................. 51 NeerAsherie PART II MEASURING PROTEIN SELF-ASSOCIATION, AGGREGATION, AND CRYSTALLIZATION 5 Integralcaa -CytochromecOxidasefromThermusthermophilus: 3 PurificationandCrystallization ........................................... 61 OrlaSlattery,SabriCherrak,andTewfikSoulimane 6 AggregationProfilingofC9orf72DipeptideRepeatProteins TransgenicallyExpressedinDrosophilamelanogasterUsingan AnalyticalUltracentrifugeEquippedwithFluorescenceDetection ............ 81 BashkimKokona,NicoleR.Cunningham,JeanneM.Quinn, andRobertFairman 7 SizeAnalysisofC9orf72DipeptideRepeatProteinsExpressedin DrosophilamelanogasterUsingSemidenaturingDetergentAgarose GelElectrophoresis ..................................................... 91 NicoleR.Cunningham,BashkimKokona,JeanneM.Quinn, andRobertFairman 8 TheUseofHighPerformanceLiquidChromatographyfor the CharacterizationoftheUnfoldingandAggregationofDairyProteins......... 103 SophieJeanneGaspardandAndre´Brodkorb 9 DifferentialScanningCalorimetrytoQuantifyHeat-Induced AggregationinConcentratedProteinSolutions............................. 117 MatthewR.Jacobs,MarkGrace,AliceBlumlein, andJenniferJ.McManus vii viii Contents 10 NanoparticleTrackingAnalysistoExaminetheTemperature-Induced AggregationofProteins ................................................. 131 SvenjaSladek,KateMcComiskey,AnneMarieHealy, andLidiaTajber 11 EvaluationofTemporalAggregationProcessesUsingSpatial IntensityDistributionAnalysis............................................ 141 ZahraRattray,EgorZindy,KaraM.Buzza,andAlainPluen 12 FluorescenceCorrelationSpectroscopyforParticleSizing inHighlyConcentratedProteinSolutions ................................. 157 JudithJ.Mittag,MatthewR.Jacobs,andJenniferJ.McManus 13 SizeDeterminationofProteinOligomers/Aggregates UsingDiffusionNMRSpectroscopy ...................................... 173 PanchamS.Kandiyal,JiYoonKim,DanielL.Fortunati, andK.H.Mok PART III COMPUTATIONAL APPROACHES TO MEASURE PROTEIN SELF-ASSEMBLY 14 PatchyParticleModelstoUnderstandProteinPhaseBehavior ............... 187 NicolettaGnan,FrancescoSciortino,andEmanuelaZaccarelli 15 ObtainingSoftMatterModelsofProteinsandtheirPhaseBehavior .......... 209 IremAltanandPatrickCharbonneau 16 BindingFreeEnergiesofConformationallyDisordered PeptidesThroughExtensiveSamplingandEnd-PointMethods .............. 229 MatthewG.NixonandElisaFadda 17 AtomisticSimulationToolstoStudyProteinSelf-Aggregation ............... 243 DenizMeneksedag-Erol andSarahRauscher Index ...................................................................... 263 Contributors IREMALTAN (cid:1) DepartmentofChemistry,DukeUniversity,Durham,NC,USA NEERASHERIE (cid:1) DepartmentofPhysics,YeshivaUniversity,NewYork,NY,USA; DepartmentofBiology,YeshivaUniversity,NewYork,NY,USA ALICEBLUMLEIN (cid:1) DepartmentofChemistry,MaynoothUniversity,Maynooth,Kildare, Ireland ANDRE´ BRODKORB (cid:1) TeagascFoodResearchCentre,Fermoy,Cork,Ireland KARAM.BUZZA (cid:1) DepartmentofPharmacyandOptometry,FacultyofBiologyMedicine andHealth,SchoolofHealthSciences,UniversityofManchester,Manchester,UK JORDANW.BYE (cid:1) SchoolofChemicalEngineeringandAnalyticalScience,TheUniversity ofManchester,Manchester,UK CESARCALERO-RUBIO (cid:1) DepartmentofChemicalandBiomolecularEngineering,University ofDelaware,Newark,DE,USA PATRICKCHARBONNEAU (cid:1) DepartmentofPhysics,DukeUniversity,Durham,NC,USA SABRICHERRAK (cid:1) DepartmentofChemicalSciences,BernalInstitute,UniversityofLimerick, Limerick,Ireland;DepartmentofBiology,UniversityAbou-BekrBelkaid,Tlemcen, Algeria DANIELCORBETT (cid:1) SchoolofChemicalEngineeringandAnalyticalScience,TheUniversity ofManchester,Manchester,UK NICOLER.CUNNINGHAM (cid:1) DepartmentofBiology,HaverfordCollege,Haverford,PA,USA ROBINA.CURTIS (cid:1) SchoolofChemicalEngineeringandAnalyticalScience,TheUniversity ofManchester,Manchester,UK ELISAFADDA (cid:1) DepartmentofChemistry,HamiltonInstitute,MaynoothUniversity, Maynooth,Kildare,Ireland ROBERTFAIRMAN (cid:1) DepartmentofBiology,HaverfordCollege,Haverford,PA,USA DANIELL.FORTUNATI (cid:1) TrinityBiomedicalSciencesInstitute(TBSI),SchoolofBiochemistry andImmunology,TrinityCollegeDublin,Dublin,Ireland ERICM.FURST (cid:1) DepartmentofChemicalandBiomolecularEngineering,University ofDelaware,Newark,DE,USA SOPHIEJEANNEGASPARD (cid:1) TeagascFoodResearchCentre,Moorepark,Fermoy,Cork,Ireland; SchoolofFoodandNutritionalSciences,UniversityCollegeCork,Cork,Ireland NICOLETTAGNAN (cid:1) CNR-ISC,UOSSapienza,Roma,Italy MARKGRACE (cid:1) DepartmentofChemistry,MaynoothUniversity,Maynooth,Kildare,Ireland ANNE MARIEHEALY (cid:1) SynthesisandSolidStatePharmaceuticalCentre,SchoolofPharmacy andPharmaceuticalSciences,TrinityCollegeDublin,Dublin,Ireland MATTHEWR.JACOBS (cid:1) DepartmentofChemistry,MaynoothUniversity,Maynooth,Kildare, Ireland PANCHAMS.KANDIYAL (cid:1) TrinityBiomedicalSciencesInstitute(TBSI),SchoolofBiochemistry andImmunology,TrinityCollegeDublin,Dublin,Ireland JIYOONKIM (cid:1) TrinityBiomedicalSciencesInstitute(TBSI),SchoolofBiochemistry andImmunology,TrinityCollegeDublin,Dublin,Ireland BASHKIMKOKONA (cid:1) DepartmentofBiology,HaverfordCollege,Haverford,PA,USA RAMILF.LATYPOV (cid:1) Sanofi,Framingham,MA,USA ix