S2004 Methods for characterization of biomolecular interactions – classical versus modern Mgr. Josef Houser, Ph.D. houser@mail.muni.cz Sufrace-based methods Interaction on surface • Interaction with • One binding partner immobilized on surface – ligand • Second binding partner free in solution – analyte • Biosensors based on various techniques • Surface plasmon resonance (SPR) • Biolayer interferometry (BLI) • Grating-Coupled Interferometry (GCI) • Quartz-crystal microbalance (QCM) • Surface acoustic wave (SAW) • Switch-sense • ... Electro-mechanical Chemo-optical Electro-chemical Mechano-optical Microcantilever Colorimetric Impedance AmperometricPhotoacoustic Piezoelectric SPR BLI History 1950’s1900’s Anomalous light reflection on metal grating (R. W. Woods) Definition of plasmon 1980 First SPR use in biomolecular interaction analysis 1990 First commercial SPR instrument (Biacore) 0 500 1000 1500 2000 2500 1982 1989 1994 1999 2004 2009 2014 2019 Papers dedicated to SPR/BLI (Pubmed) First commercial BLI instrument (ForteBio) 20081887 Invention of interferometry as a technique (A. Michelson) Surface plasmon resonance (SPR) Collective oscillation of free electrons on metal-dielectric interface SPR – Basic principles At the conditions of total internal reflexion (angle, wavelength) the incoming beam evokes exponential wave spread in opticaly less dense environment. At certain combination of incident angle and wavelength the free electrons on the metal surface are excited, what causes decrease in reflected light intensity. This effect depends on refractive index that varies with the analyte binding to the surfacebound ligand. SPR – Basic principles Refractive index change = change in light intensity at certain wavelength. Corresponds also to change of mass on the chip surface = protein/ligand binding. (1 RU ~ 1 pg/mm2) Correlation between SPR Response and Surface Concentration SPR – Basic principles One binding partner immobilized on the chip surface (ligand), second is free in solution (analyte). BLI – Basic principles One binding partner immobilized on sensor surface (ligand), second partner is free in solution (analyte). • Light reflects from the inner tip surface and outer tip surface resulting in formation of interference pattern. • Binding of analyte on the sensor tip results in change of the thickness of the optical layer -> shift in the interference pattern. BLI biosensor tip surface WAVELENGTH (nm) Biocompatible surface BLI signal processing Incident white light Optical fiber tip Reflected beams RELATIVEINTENSITY Immobilized molecule Unbound molecules have no effect λ (spectral shift due to change in thickness) Optical layer Association Dissociation     MX XM K 1 K A D === a d k k Receptor ligand interaction       MXXM MX da kk dt d −=   0 MX : = dt d mequilibriu + • Kinetics of interaction • Steady state Binding experiment v(association) = ka * [analyte](solution) v(dissociation) = kd * [analyte](bound) [analyte](solution) >> [analyte](bound) v(association) >> v(dissociation ) association phase v(association) = v(dissociation) steady state -> response is proportional to KD and Rmax [analyte](solution) << [analyte](bound) v(association) << v(dissociation ) dissociation phase Simple binding - kinetics Typical binding curve 1 - Association 2 - Dissociation 3 - (Surface regeneration) 1 2 3 3 Time s Response RU 1035 Time ResponseConcentration = 100 nM Same affinity but different kinetics • All 4 compounds have the same affinity KD = 10 nM = 10-8 M • The binding kinetic constants vary by 4 orders of magnitude kon koff M-1s-1 s-1 106 10-2 105 10-3 104 10-4 103 10-5 1035 Time Concentration = 1000 nM Completely blocked target - all target sites occupied Compounds with slow off-rates occupy the target for a longer time Same affinity but different kinetics koff (s-1) kon(M-1s-1) 104 0.0001 0.001 0.01 0.1 1 107 106 105 102 103 1 nM100 pM 10 nM10 pM 100 nM 1 M 1 mM 100 M 10 M KD On-off rate map Kinetics vs. affinity in Drug design High affinity – first aim in drug discovery BUT May be caused by high ka and kd = fast dissociation (!) Kinetics – lower ka AND kd may mean longer effect This fact is known but usually not considered ! Experiment Simple binding - kinetics Typical binding curve 1 - Association 2 - Dissociation 3 - (Surface regeneration) 1 2 3 3 Time s Response RU Chip surface Gold Matrix Protein Analyte Concentration Analyte 1 Analyte 2 Specificity 0 100 200 300 400 500 0 100 200 300 400 500 600 700 RU Response sTime Simple binding – specificity 0 100 200 300 400 500 0 100 200 300 400 500 600 700 time [s] RU 0 100 200 300 0 200 400 600 800 time [s] RU concentration concentration Ligand 1 Ligand 2 Ligand 3 Fast complex association and dissociation Fast equilibrium  KA, KD Slow complex association and dissociation Kinetic constants ka, kd  KA, KD specificity Simple binding – kinetics Simple binding – kinetics • Kinetic evaluation – fitting of association and dissociation curves Simple binding – single-cycle kinetics • Association is concentration dependent • Dissociation is concentration independent • Multiple concentration followed by single dissociation – time effective 300 800 1300 1800 -200 0 200 400 600 800 1000 1200 1400 RU Response Time s c1 c2 c3 c4 c5 Simple binding – steady state Fast association and dissociation data are not easy to fit BUT v(association) = v(dissociation) steady state -> response is proportional to KD and Rmax -20 -10 0 10 20 30 40 50 100 150 200 250 300 350 400 450 500 RU sTime Response Differential curves Fast association and dissociation KA, KD RU Response Saccharide concentration RUmax ½ RUmax [Protein] [Analyte] [Protein-Analyte] KD = RU = ½ RUmax [Protein] = [Protein-Analyte] KD = [Analyte] Direct binding assay Factors influencing binding and response • Density of the molecules on chip • Concentration of molecules in solution • Strength of interaction between both molecules • Total mass and/or way of binding of analyte • Portion of active molecules present – proper sample characterization needed, changes upon immobilization – site accessibility restriction, conformational changes, intermolecular distance • Conditions/buffer properties Sensitivity • SPR • Signal proportional to mass on the surface • High sensitive instruments – reliable analysis of <100 Da analytes (e.g. metal ions) • Suitable for both small molecules and proteins/nucleic acids • BLI • Signal proportional to thickness of surface layer • High sensitive instruments – require >1 kDa analyte or structural change of immobilized molecule • Suitable mainly for proteins/nucleic acids Which binding partner to immobilize? • Stability • Availability • Molecular mass • Immobilization technique • Multivalency Sensor Chip – rough scheme Chip surface Matrix Protein/Ligand Glass Linker layer Gold Dextran layer Specific layer User-defined biospecific surface • Biocompatible • Low non-specific binding • Robust • More than 100 runs on the same surface Immobilization techniques Direct covalent coupling • Stable • Suitable regeneration needed Capture • Multi-step process • Less stable binding • Easier regeneration (not for SA) High flexibility in creating biospecific surfaces • Amine (Lys, N-term) • Thiol (Cys) • Aldehyde • Carboxyl • Streptavidin – Biotin • NTA-Ni2+ – His6 • Anti-His – His6 • ProteinA – mAb • Anti-GST – GST Protein immobilization Chip surface Gold Matrix Protein (lectin) Saccharide N-ethyl-N’-(3-diethylaminopropyl)karbodiimide N-hydroxysuccinimide „Amine-coupling“ CM5 chip – surface modified by carboxymethylated dextran Chip surface Matrix Streptavidine Biotin Saccharide Polyacrylamide Typical spacer for saccharides is -(CH2)3-, for biotin -(CH2)6Small molecule immobilization N-ethyl-N’-(3-diethylaminopropyl)karbodiimide N-hydroxysuccinimide „Amine-coupling“ Ni-NTA utilization Activation Protein binding Regeneration +Ni2+ Sample application +EDTA Flexibility in Assay Design Multiple assay formats providing complementary data Direct measurement Indirect measurement Inhibition in solution assay (ISA) Surface competition assay (SCA)Direct Binding Assay (DBA) Time Response Inhibitor concentration Response0 % 50 % 100 % Chip surface Gold Matrix Immobilized saccharide Lectin Free saccharide Inhibition in solution assay Inhibitor concentration Response -200 -100 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 Time s Rel.response RU 0-250 μM D-mannose Inhibition of binding of B. cenocepacia lectin to immobilized D-mannose Inhibition in solution assay Inhibitor concentration Response 0-80 mM D-galactose -200 -100 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 Time s Rel.response RU Inhibition in solution assay Inhibition of binding of B. cenocepacia lectin to immobilized D-mannose 37 IC50D-mannose IC50saccharide Effectivity = Lectin from B. cenocepacia: Benzyl-α-D-mannoside ≈ Methyl-α-D-mannoside ≈ D-mannose » L-fucose > D-arabinose > L-galactose > Methyl-α-L-fucoside » D-galactose 0 % 50 % 100 % Inhibition in solution assay Two channels necessary - reference • “Non-interacting” surface serves as a blank • Elimination of non-specific interactions • Enhancement of weak interaction resolution • Possible reference surfaces: • Unmodified surface – gold, dextran layer,… • Activated and blocked surface without immobilized ligand/protein • Inactivated/non-functional ligand/protein Two channels necessary - reference -50 0 50 100 150 200 250 -50 0 50 100 150 200 250 300 Time s Response RU Reference channel Multichannel set-up • One or more references • Multiple channels – 2, 4, 6, 36,… • Multiple detection spots ➢High throughput ➢Parallel reference Specialized techniques • Membrane proteins • Multi-layer approaches – Ab’s, protein complexes • Whole cell immobilization • Thermodynamics measured by SPR • Ligand recovery On-surface reconstitution approach • A very quick and easy method for functional reconstitution of immobilized membrane proteins with lipids. • Conventional immobilization techniques are applicable on membrane proteins. • Surfaces with high density of receptor can be prepared. • The lipid matrix can be renewed after every cycle. • “Lipid bilayers” can be very rapidly and easily built and rebuilt on Pioneer Chip L1 (Biacore). Immobilize a GPCR-specific mAb on a L1 chip. On-surface reconstitution approach Capture a detergent-solublized GPCR on the immobilized mAb surface. On-surface reconstitution approach Reconstitute a lipid bilayer around the receptor Inject lipid/ detergent mixed micelles across the surface. On-surface reconstitution approach Wash the surface with bufferDissociates the detergent from the micelles. On-surface reconstitution approach Establish the integrity of the reconstituted GPCRUse conformationally sensitive anti-bodies. On-surface reconstitution approach Study the kinetics of ligand/receptor interactions On-surface reconstitution approach Binding of the chemokine SDF1 to the reconstituted CXCR4 receptor -5 0 5 10 15 20 25 30 35 40 45 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (s) Response(RU) Time (s) Response(RU) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05 [SDF1] (M) Response(RU) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05 [SDF1] (M) Response(RU) -5 0 5 10 15 20 25 30 35 40 45 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (s) Response(RU) Time (s) Response(RU) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05 [SDF1] (M) Response(RU) -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05 [SDF1] (M) Response(RU) KD = 288 ± 11 nM On-surface reconstitution approach Proteomics Study BIACORE “Classical Applications” Kinetics, Affinity Structure-Activity studies Receptor Express 6-24 months Recovery Enzymatic digestion HPLC separation MS Ligand Fishing SPR/MS Main SPR biosensors • GE Healtcare – Biacore S200, Biacore T200, Biacore 4000, Biacore 3000, etc. • Reichert – SR7000DC • BioRad – ProteOn™ XPR36 • Biosensing Instrument – Bi4000, Bi3000, etc. • Nicoya – Alto, OpenSPR Biacore S200 SR7000DC ProteOn™ XPR36 Bi4000 Alto High-throughput SPR Biacore 8K (highest model) • 16 channels • Up to 4x384 samples in a run • 2300 interacting molecules/day • 64 kinetic characterizations/4 hrs www.cytivalifesciences.com Main BLI biosensors • Fortebio – BLItz, Octeet R2, Octet R4, Octet RED96e, etc. • Gator Bio – Gator BLItz Gator Octet RED96e Octet R2 High-throughput BLI Octet HTX • Up to 96 samples simultaneously • 96 samples quantitation/2 mins • Up to 32x32 epitope binning/8 hrs analytica-world.com Kamat 2017 Objectives of SPR/BLI experiment oKinetic Rate Analysis: How FAST? • ka, kd • KD = kd/ka, KA = ka/kd o Yes/No Data • Screening • Ligand Fishing o Concentration Analysis: How MUCH? • Active Concentration • Solution Equilibrium • Inhibition o Affinity Analysis: How STRONG? • KD, KA • Relative Ranking On-surface technology advantages • No labeling • Real-time • Unique, high quality data on molecular interactions • Simple assay design • Robust and reproducible • Walk-away automation • Small amount of sample required Method comparison SPR BLI MST ITC AUC Parameters KD/KA, kon, koff KD/KA, kon, koff KD/KA, N KD/KA, N, ΔG, ΔH, ΔS KD/KA, N KD range [M] 10–13 – 10–3 10–11 – 10–3 10–11 – 10–1 10–12 – 10–2 10–8 – 10–4 Speed (per KD) 15 – 120 min 15 – 60 min 15 – 30 min 30 – 120 min 4 – 72 hod Sample modification Immobilization Immobilization Labeling None None Complex samples ✓ ✓ ✓   High throughput ✓ ✓ ✓ ✓  Materials for further study http://www.sprpages.nl/ Materials for further study Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz CF Head: Michaela Wimmerová • +420 549 498 166 • michaela.wimmerova@ceitec.cz bic@ceitec.cz bic.ceitec.cz Biomolecular I nteraction and Crystallization Core Facility