...

Lab Projects Proster

by user

on
Category: Documents
1

views

Report

Comments

Transcript

Lab Projects Proster
Pharmaceutical Nanoimaging and Nanoengineering
Yuri L. Lyubchenko
DepartmentofPharmaceu0calSciences,UniversityofNebraskaMedicalCenter,
986025NebraskaMedicalCenter,Omaha,NebraskaUnitedStates68198
Email:[email protected]
Overview
§  Renewable energy technologies provide an environmentally clean and efficient source of energy.
Researchinthelabfocusesonunderstandingfundamentalmechanismsunderlyinghealthanddisease,which
arekeytodevelopingnewandmoreeffec1vediagnos1csandmedica1ons.Thisprimarilybasicresearchallows
usnotonlyiden1fiesnewdrugtargetsforsmallmolecules,italsodevelopsthetoolsandmethodstodiscover
novelapproachesfordiagnos1c,treatmentanddiseasepreven1onandtomorerapidlydeterminetheir
efficacyatthemolecularlevel.
Thelabprojectsarecategorizedintofourbroadareas,withinterplayandoverlapamongthem:
•  Structuralgenomicsinrela?ontocancerandotherdiseasesNSF:MCB1515346andNIH–R01GM100156
•  MolecularmechanismsAlzheimer’s,Parkinson’sandotherproteinaggrega?ondiseases-NIH–R01GM096039
•  Protein-DNAinterac?onsfornovelapproachesforHIVrestric?on–NIHR01GM118006
•  Developmentofnovelnanoimagingandnanoprobingapproaches–allNIHandNSFgrants
UNMC
Area3:protein-DNAinterac1onsanddevelopmentofapproachesforHIVrestric1on
IntheareaofHIVrestric?on,weareworkingonmolecularmechanismofinterac?onwithDNAAPOBEC3proteinsthatconsidered
asinnateimmunesystemagainstHIV.WedevelopedadvancedexperimentalAFMapproachesthatallowedustocharacterize,at
thenanoscale,complexesformedbyA3GHIVrestric?onfactorandssDNA.Thesetechniquesenabledustodirectlyimagethe
slidingofA3Galongthesubstrate.Further,wediscoveredandcharacterizedtheprocesswhereA3Gself-assemblesintooligomers.
Together,thesestudiesdeterminedanumberofnovelfeaturesofA3G-ssDNAcomplexes,thuspavingtheroadfornew
therapeu?cinterven?ons.
Sliding of A3G. Selected
frames for A3G-gap-DNA
complex (a) and gap-DNA
(b).
Barsize50nm.
Each frame 720 msec.
Ver?caldoeedlinesshow
the distance between
endsofDNAarmsandthe
centeroftheprotein.
Area 1: chromatin structure and dynamics
Inthechroma?narea,wediscoveredhighlydynamicinherentpropertyofnucleosomesbasedonthedirect
imagingofnucleosomeswith?melapseAFMandiden?fiedacri?calroleofelectrosta?csintheirspontaneous
dynamics.Thecurrentprojectdealswithcharacteriza?onofcentromerechroma?n.
Thenucleosomeunwrappingprocessimagedwith?melapseAFM.(A)Consecu?veAFMimagesofnucleosomes
withatwo-stepunwrappingprocess,takenduring
con?nuousscanninginthebuffer.Eachframeis200nm.(B)
Dependenceofarmlengthsontheframenumber.
Ourcurrentmodel:Schema?cstructuresofH3andCENP-A
mononucleosomes(aandb,respec?vely)andtheir
arrangementsintoarrays(candd,respec?vely).Histonecores
areshownasyellowandgreencircles(Lyubchenko,Nature
Struct.Mol.Biol).
Area 1: DNA replication. Molecular mechanism of the reversal of the replication fork regression
(A)AFMimagesofthecomplexoftheDNArulerwithA3G
proteinformedinthepresenceof5mMMg2+.Blackandblue
arrowsindicatecomplexeswithdimersandmonomersofA3G
respec?vely.Greenarrowheadsarepointedtofreeproteins.(B)
Histogramofthevolumemeasurementsforthecomplex.(C)The
volumemeasurementsforthefreeprotein.
Area 4: Technology development. Thetechnologydevelopmentareahasalwaysbeenanintegral
partofallofallresearchprojects.Notethesampleprepara?onproceduredevelopedforAFMimagingofDNA,RNA
andprotein-nucleicacidsystemthatmadeitpossibletodirect?me-lapseimagingofvariousnucleoprotein
complexes.
NovelsilatranesurfacechemistryforAFMimaging
andsinglemoleculemanipula1on
•  Regression of a stalled fork proceeds from left to right,
•  Reversal proceeds from right to left restoring the original fork. Alternatively, the central toe could be degraded
(dashed lines) up to the junction, restoring the fork (right).
•  RecG helicase accomplishes the regression process.
AFMimagesandModelsforRecGbindingtotheDNAforkinthepresenceofSSB.
(A)–nointerac?onwithSSB.Intype1complexRecGbindsspecificallytoallarmsoftheforkinwhichdomains1and2bindthe
parentalDNAduplex,whereaswedgedomain3bindssingle-strandedarmofthefork.SSBoccupiesthessDNAarmofthefork.(B)
Intype2complexformeda]ertheSSBremodeling,thewedgedomain3dissociatesfromthessDNAarm,butdomains1and2
remainboundtotheparentalDNAflank,enablingRecGtotranslocatealongtheduplexasshownin(i)and(ii).Model(i)showsthe
RecGposi?onattheforkjusta]erre-modelingand(ii)showsRecGposi?ona]ertransloca?onawayfromthefork.
LabPage
Noaggrega?ononthesurface
Lowsaltsolu1on
AFMofcruciformsShlyakhtenkoetal
(1998)JMolBiol
Highsaltsolu1on
CruciformsandZ-DNA
Lushnikovetal(2004)NAR
LowpH
AFMofH-DNA
Tineretal(2001)JMolBiol
Area 2: molecular mechanisms Alzheimer’s, Parkinson’s and similar protein misfolding diseases
High-speedAFM–T.Ando,
KanazawaUniversity
In the protein misfolding area we have made fundamental
contribu?ons to understanding molecular mechanisms of such
neurodegenera?ve disorders as Parkinson’s, Alzheimer’s, and
Hun?ngton’s diseases. We developed novel approaches for
probingproteinsmisfoldedstateswithAFMforcespectroscopy.
Themajordiscoveryisthatamisfoldingamyloiddimerhasan
extremely long life1me in comparison with the fast
conforma1onal dynamics of monomers. Such stabiliza?on
drama?callyfacilitatestheaggrega?onkine?csandthustriggers
the en?re aggrega?on process. This conclusion is in line with
recentfindingsonthecri?calroleofdimersintheself-assembly
of proteins and supported by computa?onal approaches in
which we u?lize na?onal most powerful computa?onal
resources.
AFMimagesofself-assembledRNAnanostructures
Directobserva?onofthe
DNAcleavagewithhighspeedAFM
Schema?cofAFMforcespectroscopy
experimentusedtomeasuretheinterac?onsof
moleculesimmobilizedontheAFM?pand
surfaceviaflexibletethers.
Aschema?crepresenta?onoftheprotein
dynamics.Thetransi?onsofthefolded(na?ve)
stateoftheproteintounfoldedandmisfolded
statesareshownforthesimplicityonly.
Characteris?c?mesforthetransi?onbetween
thesestatesaswellasthedimerlife?meare
shownabovethearrows.
Current lab members
• 
• 
• 
• 
• 
Luda Shlyakhtenko
Zhengjian Lv
Siddhartha Banerjee
Zhiqiang Sun
Micah Stumme-Diers
• 
• 
• 
• 
• 
Sibaprasad Maity
Jamison Duckworth
Mohtdin Hashemi
Yangang Pan
Galina Warren
Fly UP