Harnessthepowerofintracellularcompoundstoimproveyourcrystallisations.
ThenewDurhamOsmolytescreenallowsyoutotestwhichofabroadrangeofcellcomponentshelpstABIliseyourprotein,improvingtheresultsofstructuralandfunctionalstudies.ThedevelopersofthescreendescribetheimportanceofstabilityscreeningforproteinstructuralandfunctionalstudiesinthisJoVEarticle:
Osmolytesareuncharged,highlysolubleorganicmoleculesthatactincellstobalancehighexternalsaltconcentrationswithoutdisturbingintracellularionicstrength.Thismakesosmolytestheidealadditivesforstabilisingyourproteinduringstructuralandfunctionalstudiesastheyareusuallypresentinhighconcentrationsinnativecellenvironmentswithoutaffectingproteinbehaviour.
EasilyidentifywhichosmolytesaremostbeneficialtoyourresearchwithTheDurhamOsmolytescreen,a96-wellscreenthatscansmorethan50differentosmolytes,availableexclusivelyfromMolecularDimensions.
Osmolytessuchassugars,polyols,amino-acids,NDSBsandmanyotherchemicalclassesareincludedinthescreen,forexample:
Tooptimiseyourprotein’scompleteenvironment,werecommendusingtheOsmolytescreentogetherwiththeDurhampHandSaltscreens.AllthreescreensweredevelopedatDurhamUniversitybyEhmkePohl,DanielBruce,MortenGrøftehaugeandEmilyCardew.
TheDurhamScreensaredesignedtoworkwithNAMI*thefreetodownloadsoftwareforeasyinterpretationofthermalshiftassayresults.Reference:Bruce,D.etal.J.Vis.Exp.144:e58666(2019).TheDurhamOsmolyteScreenispresentedas96x0.5mLconditionsinadeep-wellblock.Theyarepreservativefreeanditisrecommendedthattheyarestoredat4°C,howevertheycanbeusedatroomtemperatureforscreenset-up.*NAMIcangeneratewaterfallplotsofthermalshiftdataatmultipleconcentrationsorplotsofmeltingtemperatureagainstconcentration.Thesedifferentiateasuddenincreaseinproteinstabilityindicatingaspecificinteractionandsteadyincreasesinstabilityduetothechangingproteinenvironment.Inaddition,itproducesausefulcolourgrADIentplatediagramtomakeidentifyingparticularlystabilisingordestabilisingconditionssimple.ThermoFluorisaregisteredtrademarkofJohnsonandJohnson.
