New ways to explore biomolecular interactions 1 Outline
New ways to explore biomolecular interactions 1
Outline n Biomolecular interactions background n Three new techniques to characterize biomolecular interactions q How BITC has played a role mac 122. icu. ac. jp/gen-ed/cell-division. html 2
Interactions and solution nonideality > 1. 0 repulsion ~ 1. 0 ideal < 1. 0 attraction Solute-solvent Solute-solute Solvent-solvent 3
Potential energies affecting γ + + - < 1. 0 > 1. 0 4
Potential energies, strength and distance 5
Techniques for characterizing interactions Binding and separation n q n n Gel shift Footprinting Cross linking Chromatography q n n q CD Fluorescence/depolarization Phenomenological q n n Osmometry Light scattering Mass spectroscopy SPR/TIRF NMR, ESR Size, affinity Two-hybrid assays Optical spectroscopy q n Filter assays, dialysis n n Analytical electrophoresis Analytical ultracentrifugation Calorimetry Activity change, etc. 6
Analytical electrophoresis Determine the net charge (valence) on molecules n n n Membrane confined electrophoresis Capillary electrophoresis Electrophoretic light scattering (not yet) 7
Accurate valence determination n Determine z* n Convert z* z. DHH Durant JA, Chen C, Laue TM, Moody TP, Allison, SA (2002) “Use of T 4 Lysozyme Charge Mutants to Examine Electrophoretic Models” Biophys. Chem. , 101102: 593 609. Moody TP, Shepard HK (2002) “Nonequilibrium Thermodynamics of Membrane. Confined Electrophoresis” Biophys. Chem. 108: 51 -76 8
Valence accuracy Sue Chase/BITC 9
Ig. G 100 m. M KCl, 10 m. M MES p. H 6. 0 MAb ZCalc ZDHH GE k. J/mole A 24 2 +16 B 32 13 +25 C 36 16 +32 Sue Chase/BITC 10
m. Abs charge differs from expectations 100 m. M KCl 10 m. M MES p. H 5. 0 Z < Zcalc by 51 100 m. M KCl 10 m. M MES p. H 6. 0 Z < Zcalc by 17 Sue Chase/BITC 100 m. M KCl 10 m. M buffer 11
Whole MAb versus Fc and Fab 100 m. M KCl 10 m. M MES p. H 6. 0 m. Ab p. I Zcalc Peak % ZDHH (SD) Whole 8. 01 32 1 72. 1 7. 1 (0. 44) Whole 8. 01 32 2 27. 9 11. 9 (1. 43) Fab 7. 86 7. 4 1 100 4. 0 (0. 28) Fc 7. 14 18. 2 1 20. 6 0. 4 (0. 23) Fc 7. 14 18. 2 2 65. 5 1. 0 (0. 49) Sue Chase/BITC 12
MAK 33 m. Ab FAb showing predicted electrostatic map n BITC collaboration using MAK 33 q Intact m. Ab & fragments n q n Glycoslyated versus deglycosylated Undergraduate Mc. Nair fellowship q n FAb, CL, VH, B 2 M Mai Doan- Charge and interacting systems Graduate Wyatt fellow q John Champagne- aggregation and phase separation of MAbs 13
Membrane confined electrophoresis apparatus n Gold standard for accurate charge determination Higher accuracy than CE n Spin Analytical n q Available to BITC member companies 14
Analytical ultracentrifugation NUTS & BOLTS n n n First principle method for characterizing solution interactions New tricks for an old dog Advancing methods for complex, concentrated solutions 15
AU-FDS BITC 40 n. M GFP in 100 m. M KCl, 10 m. M Tris, p. H 8. 0 Spatial filter Collimation Laser PMT Dichroic filter Dichroic mirror Expansion Objective lens Fluorescent event 100 n. M GFP 100 n. M Anti GFP in Human serum 40 n. M GFP in E. coli lysate 16
NUTS: High affinity interaction F-r. TTR in 100 m. M KCl, 10 m. M Tris, p. H 9 + 0. 1 mg/ml ovalbumin Jon Kingsbury 17
NUTS: Oligomer stability modified protein 100 m. M KCl, 10 m. M Tris, p. H 9 + 0. 1 mg/ml ovalbumin F-r. TTR-SO 3 □ F-r. TTR ○ F-r. TTR-cys ∆ Jon Kingsbury 18
NUTS: Effect of drug F-r. TTR in 100 m. M KCl, 10 m. M Tris, p. H 9 + 0. 1 mg/ml ovalbumin F-r. TTR □ 1 mg/ml drug 1% DMSO F-r. TTR ○ No drug 1% DMSO Jon Kingsbury 19
NUTS: Sedimentation equilibrium of a high-affinity interaction 25 n. M 6. 3 n. M 1. 6 n. M 0. 39 n. M Co-analysis of 10, 000 & 16, 000 rpm data yields Kd = 1. 6 n. M Burgess, et al, (in press) “Structure and evolution of a novel dimeric enzyme from a clinically-important bacterial pathogen” J. Biol. Chem. , Aug 2008 20
BOLTS: concentrated complex solutions n n Weak forces are important Adopt statistical concept of “binding” q Correlation of motion = “binding” nγ<1 q Anti-correlation = “nonideality” nγ>1 n Aviv AU-FDS provides γ q γ = capp/c = M/Mapp 21
BOLTS: GFP-labeled construct cell lysate Sue Chase 22
Repulsion BOLTS: concentrated solutions STI Dextran HEL Attraction Rachel Kroe/BITC 23
BOLTS: Immuno-sedimentation Kari Hartman/BITC 24
BOLTS: Ig. G in serum 500 n. M Alexa in TBS or human serum ΔS ≈ 1. 7 BSA Complex Rachel Kroe 25
BOLTS: Immuno-sedimentation in Serum GFP-anti. GFP in serum GFP Rachel Kroe Ig. G 26
Innovations for AUC A new cottage industry • Al 7075 cell components • Quantity discount windows • Cell inspection/refurbishing • Improved torque stand • 1. 2, 3, 12 mm pathlength cells • Spin Analytical • Aviv Biomedical • Nanolytics- http: //www. spinanalytical. com http: //www. avivbiomedical. com http: //www. nanolytics. de 27
The Open AUC Project Buffer Chooser Analyte Chooser Instrument Control Software (AU-AOS) commands data Non-Interactive Analysis Software G(s), match, etc. User Interactive Analysis Software Client Library Sednterp 2 XML data PANDa. S data Legacy Data Store Sedfit, Sedanal, etc. XML results Access Control Database Subscriber List Database Library Analysis Results Analyte Data Experiment Results Buffer Data 28
Open AUC: Spin Analytical CFA n Open AUC platform q q q n Open software q q n AOS machine socket + XML-RPC PANDa. S compliant Encourage 3 rd party developers q q n Optics outside of vacuum chamber 8 -hole, 60 K rotor Same cells, windows Open architecture bus + drivers Interfaces to ease new optics development Detectors q q q Rapid scan absorbance Multi-wavelength absorbance Low angle light scattering Multi-wavelength fluorescence Interference Schlieren 29
Plasma differential scanning calorimetry BITC: Brad Chaires, U. Louisville n n First principle method for determining energies Mixtures resolved with respect to their thermal stability (enthalpy, Tm) q n Rather than their size, shape or charge Sensitive to the extensive properties of the system q The mass concentration of each component. 30
>3000 proteins in the plasma proteome Ten proteins comprise 90% of plasma by weight; another twelve 9% more Anderson, N. L. & Anderson, N. G. (2002) Molecular & Cellular Proteomics 1. 11: 845 -867 Anderson, N. L. et al. (2004) Molecular & Cellular Proteomics 3. 4: 311 -326 31
Protocol 32
DSC for 15 “normal” individuals 33
DSC of diseased individuals 34
DSC of diseased individuals 35
Average of diseased individuals, N = 5 - 12 36
Origin of Shifts in Thermograms: Evidence in support of the “interactome” hypothesis? BG = Bromocresol Green; Binds to HSA Site I with K=7× 105 M-1 37
Complementary BITC project n A Disposable Polymeric Microdevice for Energetic Characterization of Biomolecular Interactions q q n n DSC on a chip BITC project 2006. 1 Qiao Lin, Columbia University 2009 BITC roundtable on micro-DSC 38
BITC: New instruments and methods Present n Disposable DSC on a chip q n Calorimetric drug-plasma protein interactions q n n Gryczynski, UMBC, U. N. Texas Src-containing complexes in cancer cells q n Hayes, BBRI Fiber optic SPCE q n Neubig, U. Michigan Sednterp 2 q n Chaires, U. Louisville Quantitative multiplexed flow cytometry interaction assay q n Lin, Columbia U. n Fluorescence detected sedimentation n Surface plasmon-coupled emission fluorescence n Fluorescence methods for protein interaction analysis n Real-time analysis & display of AUC data n High-throughput absorbance AUC q q q n n Laue, UNH Stafford & Hayes, BBRI Laue, UNH Myszka, U. Utah Laue, UNH Dynamic force measurements on AFM q n Correia, U. Miss. Med. Sch. Testing electrophoretic light scattering for z determination q n Gryczynski, UMBC, U. N. Texas Testing and validation of PWR q n Laue, UNH GPCR reconstitution & capture on SPR surface q Villari, St. Anselm College m. Ab valence m. Abs in Serum q Past Moy, U. Miami Quantitative FRET analysis of protein-protein interactions in cells q Christensen, Clemson U. 39
BITC- who are we? 40
What do we do? n n n Educational programs Develop instruments and methods for characterizing molecular interactions Evaluate new instruments and technologies 41
Educational programs n Workshops q q n Roundtable discussions q q n 2007: Physical properties of m. Abs 2008: Reform school for ill-behaved proteins 2007: Nanocalorimetry 2008: Comparability studies Online Technical Resource Center q Instrumentation, methods, software, tutorials, references, links 42
New instruments and methods Present n Disposable DSC on a chip q n Calorimetric drug-plasma protein interactions q n n Gryczynski, UMBC, U. N. Texas Src-containing complexes in cancer cells q n Hayes, BBRI Fiber optic SPCE q n Neubig, U. Michigan Sednterp 2 q n Chaires, U. Louisville Quantitative multiplexed flow cytometry interaction assay q n Lin, Columbia U. n Laue, UNH Fluorescence detected sedimentation q n n Stafford & Hayes, BBRI High-throughput absorbance AUC q n Correia, U. Miss. Med. Sch. Real-time analysis & display of AUC data q n Gryczynski, UMBC, U. N. Texas Fluorescence methods for protein interaction analysis q n Laue, UNH Surface plasmon-coupled emission fluorescence q Laue, UNH GPCR reconstitution & capture on SPR surface q Myszka, U. Utah n Testing and validation of PWR n Testing electrophoretic light scattering for z determination n Dynamic force measurements on AFM n Quantitative FRET analysis of protein-protein interactions in cells q Villari, St. Anselm College m. Ab valence m. Abs in Serum q Past q q q Myszka, U. Utah Laue, UNH Moy, U. Miami Christensen, Clemson U. 43
Acknowledgements Rachel Kroe Brett Austin Sue Chase Jon Kingsbury BITC CAMIS NIH NSF Jeff Hansen Borries Demeler Edward Eisenstein Jack Aviv 44
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