Center for Center for Macromolecular Interactions Macromolecular Kelly Arnett, Ph.D. Interactions

What is the CMI? Surface Plasmon Resonance (SPR) Size-exclusion Chromatography with Multi-angle Light Scattering The BCMP Center for Macromolecular Interactions (CMI), is a biophysical SPR is a technique to measure biomolecular interactions between an immobilized ligand (SEC-MALS) instrumentation facility for the characterization of macromolecules and their and an analyte in solution. SPR can occur when plane-polarized light hits a metal film Using a multi-angle static light scattering (MALS) detector, the intensity of scattered light interactions. The CMI mission is to enhance basic research in the HMS community under total internal reflection conditions. The SPR signal is directly dependent on the can be used to measure a weight-average molar mass in solution. Size-exclusion by providing scientific consultation, training and access to shared biophysical refractive index of the medium on the sensor chip. The binding of biomolecules results chromatography (SEC) separates molecules based on hydrodynamic volume. equipment. The facility currently includes instruments for Isothermal Titration in changes in the refractive index on the sensor surface. Real-time measurements of Combining SEC and MALS in an SEC-MALS experiment allows for more accurate mass Calorimetry (ITC), Surface Plasmon Resonance (SPR), Biolayer Interferometry binding allow determination of association and disociation rate constants (ka and kd) and measurements that either method alone. Combining two concentration detection (BLI), MicroScale Thermophoresis (MST), Differential Scanning Fluorimetry equilibrium binding constants (KD). modes (UV and RI), conjugate analysis can be performed to determine the mass Biacore T200 (DSF), (CD), and Analytical Size Exclusion Chromatography with contribution of modifiers such as carbohydrate or detergents. Multi-Angle Light Scattering (SEC-MALS). a b detector Polarized Kinetic Analysis

Reflected Intensity light ∝ ・ ・ 2 6 light 40 laser I (θ) M c (dn/dc) 1×10 1.0 w Normalized Refractive Index kon prism a b θ glycan sensor chip angle 30 What can you do at the CMI? total A + B AB gold film 2 5 • koff b Fit: ∑ niMi 1×10 Measure Molecular Interactions RU 20 time flow chamber ka, kd Mw= - A B koff buffer ∑ = a 10 niMi 4 KD KD = 1×10 0.5 - Nucleic Acids AB kon a 0

M = 50 Molar Mass (Da) - time flow chamber Resonance signal 0 100 200 300 400 w Small molecules Binding Equilibrium Binding Kinetics sample 3 b time time [s] 1×10 1.0 Dissociation 99 Association • Characterize Protein Properties Phase Dissociation 2 0.0 Phase Phase Steady-state Analysis Single-cycle Kinetics 1×10 analyte 40 60 1 18 19 20 21 - 0.5 Association Secondary Structure [AB] flow Phase sample ∝ fraction bound Wyatt time [min] Response fraction bound response - Thermal Stability (Tm) Regeneration 30 complex high mannose shaved baseline baseline 40 fit Fit: K Dawn M = 1 - 0.0 response D w Mw = 99 (J. Li, T.A. Springer)

Mass and Oligomeric State RU load RU 20 KD concentration time Kd baseline Heleos II ligand concentration, [L] off on 20 baseline 10 immobilized ligand 0 0 buffer Load buffer analyte buffer regeneration buffer 0.5 1.5 2.5 3.5 0 500 1000 1500 Circular Dichroism (CD) sample sample buffer CMI Technologies for measuring binding concentration [µM] time [s] time CD is a spectroscopic method for determining the optical isomerism of Size Exclusion Isothermal Differential (R. Pascolutti, A, Kruse) Surface Plasmon MicroScale Chromatography molecules. Circular dichroism (measured in molar ellipticity) is the difference in Biolayer Interferometry Titration Scanning technology Resonance Thermophoresis with Multi-Angle (BLI) Calorimetry Fluorimetry absorption of left-handed and right-handed circularly polarized light and can be (SPR) (MST) Light Scattering (ITC) (DSF) Bio-Layer Interferometry (BLI) (SEC-MALS) observed in molecules with chiral centers. CD spectra in the "far UV" region (185-250 NanoTemper BLI is an optical technique for measuring macromolecular interactions by analyzing ForteBio ForteBio GE Microcal Technologies Life Technologies Wyatt nm) can be used to determine protein secondary structure. Thermal stability (Tm) can instrument interference patterns of white light reflected from the surface of a biosensor tip. A Octet RED384 BLItz Biacore T200 ITC200 Monolith Quant Studio 6 Dawn Heleos II be measured by following changes in molar ellipticity with increasing temperature. NT.115pico change in the number of macromolecules bound to the end of the biosensor tip causes Change in Change of interference pattern of Change of Thermophoresis Scattered light Enthalpy of protein-binding a shift in the interference pattern that can be measured in real-time to determine CD Scan CD Melt signal white light due to size of bound refractive index induced change intensity during binding dye fluorescence molecule due to mass in fluorescence separation intensity association and dissociation rate constants (ka and kd) and equilibrium binding constants Tm, yes/no MW, n, yes/no (KD).

measures ka, kd, KD ka, kd, KD ΔH, ΔS, n, KD KD, EC50 binding, pseudo- dmol 40 Kinetic Analysis / binding 0.3 2 KD Δλ Jasco J-815 cm

a b ・ Elipticity

0.2 Fit: ForteBio deg KD range

circularly polarized Molar ka, kd 0 BLItz x10 ]

0.1 θ [ Relative intensity sample and protein sample, Depends on SEC Relative intensity light Relative intensity

sample/analyte analyte analyte response (nm) analyte - - non-protein capacity limits >200 Da >150 Da 0.0 β-sheet >10,000 Da analyte (>5,000 Da) Wavelength (nm) Wavelength (nm) Wavelength (nm) 0 50 100 -40 α-helix 80-220 µl per 20 µl per well time [s] sample volume ~5 x 4 µl per ~200 µl per ~300 µl per 10 µl per capillary white white 200 220 240 measurement (up (perform 2-4 5-100 µl per run light light wavelength (nm) per experiment measurement immobilization titration (16/experiment) to 16 at once) replicates) Steady-state Analysis b (Adapted from N. Greenfield, 1969) (A.Schoen, 2013) 0.3 ~ 100-200 µg

sample conc. 10-50 µg/ml 10-50 µg/ml 5-50 µg/ml 10x KD, >5µM 50 pM-µM 0.05-5 µg/well (nm)

(varies by MW) λ 0.2 Δ 80-220 µl for 4 µl for each of ~300 µl for each 70 µl for each Differential Scanning Fluorimetry (DSF) 20 µl per (mixed with (mixed with a Fit: K analyte volume each of ~5 ~5 of ~5 titration 0.1 D experiment sample) sample) response (nm) concentrations concentrations concentrations (~140 µl/expt) time DSF uses a real-time PCR instrument to monitor thermally induced protein denaturation buffer sample 0.0 ≥ sample, ≥ sample, well well 0 50 100 150 200 by measuring changes in fluorescence of a dye that binds preferentially to unfolded analyte conc. 0.1-10 KD 0.1-10 KD 0.1-10 KD ~100x KD ≥ 40x KD (Adapted from ForteBio) Concentration [µM] >10x KD >10x KD ForteBio Octet RED384 protein (such as Sypro Orange, which binds to hydrophobic regions of proteins exposed by unfolding). This experiment is also known as a Protein Thermal Shift Assay, because MicroScale Thermophoresis (MST) Isothermal Titration Calorimetry (ITC) shifts in the apparent melting temperature can be measured upon the addition of MST is an immobilization-free technology for measuring biomolecular interactions ITC is a label-free method for measuring binding of any two molecules that release or stabilizing or destabilizing binding partners or buffer components. ) with a wide range of affinities (pM-mM). The MST instrument detects the motion of absorb heat upon binding. ITC monitors heat changes by measuring the differential fl Life Technologies fluorescent molecules along a microscopic temperature gradient, which reflects power, applied to the cell heaters, required to maintain zero temperature difference Folded QuantStudio 6 changes in the molecular hydration shell, charge or size. Since one or all of these between a reference and a sample cell as the binding partners are mixed. ITC can be protein parameters changes with virtually every binding event, a wide range of biomolecules used to measure the thermodynamic parameters of biomolecular interactions, Fluorescence ( Fluorescence can be measured from ions and small molecule fragments to large macromolecular including affinity (KA), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n). Fluorescence (Fl) complexes in very small volumes (<10 ul) in a wide range of standard buffers and + Protein complex mixtures including liposomes, detergent, serum, and cell lysates. A B C Protein + Ligand ΔG = RT lnKD 10 Hydrophobic dT injection protein dye )/ 1 2 3 4 fl infrared laser non-fluorescent syringe unfavorable d(

ΔG d(Fl)/dT ligand ΔG = ΔH -TΔS 0 excitation ΔH 1.0 light Temperature Laser On kcal/mol -TΔS 0.9 1 Time (min) -10 30 40 50 60 70 80 4 objective favorable 0 30 60 90 0.8 fluorescent TemperatureTemperature 2 capillaries target reference sample 0 0.7 -20 3 cell cell 0.6 Normalized Fluorescence Normalized cal/s -2 0.5 µ 0 10 20 scanning Time [s] direction Contact Visit The CMI is supported by -4 1.00 0 Harvard Medical School the BCMP Department. 650 Microcal Kelly Arnett, Ph.D. affinity: KA=1/KD Kd = 63 nM -5 240 Longwood Avenue HMS Tools and Technology, Ligand Director enthalpy: ΔH ITC200 and user access fees. Norm

0.75 600 mol Building C, Room 303 F -10 [email protected] Kd = 4.0 µM stochiometry: N Boston, MA 02115 kcal/ -15

Normalized Fluorescence Normalized 550 0.0 0.5 1.0 1.5 2.0 0.50 NanoTemper -5 0 5 10 15 20 25 0.001 0.01 0.1 1 10 100 Molar Ratio (ligand/protein) Time [s] concentration [µM] Monolith NT.115pico (R.Behrouzi, D.Moazed) cmi.hms.harvard.edu