Biochemical methods II cloning Is my protein correctly folded? Is it thermally stable? Is it a functional protein? expression purification our objectives Hongmin Tu, BCO Protein Analysis Core Facility 10/10/2018

Biophysical analyses Circular Dichroism Spectroscopy (CD): protein secondary structure melting temperature (Tm) Isothermal Titration Calorimetry (ITC): ligand binding affinity binding enthalpy stoichiometry. Surface Plasmon Resonance (SPR): binding affinity binding kinetics

I. CD spectrometry left-handed circularly polarized light (L-CPL) right-handed circularly polarized light (R-CPL) Circular dichroism (CD) = ΔA(λ) = A(λ)L-CPL - A(λ)R-CPL, λ is the wavelength The measured molecule should contain one or more chiral chromophores (light-absorbing groups): peptide, protein, RNA, DNA, etc.

CD spectrophotometer

CD measurements

Protein secondary structures

CD applications Secondary structure measurements. Detect changes in the structure of a molecule, induced by changing temperature, pH, ligands, or denaturants.   Compare two macromolecules, or the same molecule under different conditions. if a newly purified protein is correctly folded. if a mutant protein has folded correctly in comparison to the wild-type. If a biopharmaceutical product is in a correctly folded active conformation from batch to batch.

CD examples wild type protein mutant protein

CD technical limitation Advantages In solution. Very few consumables (nitrogen gas, UV-lamp). … Disadvantages Concentration: 0.02-1 mg/ml, ≥200 µl. Buffer restriction: many common buffers are not good for <200 nm measurement. Difficult for high throughput.

References Protein Circular Dichroism Data Bank: http://pcddb.cryst.bbk.ac.uk/ Whitmore L, Wallace BA (2008). "Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases". Biopolymers 89 (5): 392–400. doi:10.1002/bip.20853. PMID 17896349. Greenfield NJ (2006). "Using circular dichroism spectra to estimate protein secondary structure". Nature protocols 1 (6): 2876–90. doi:10.1038/nprot.2006.202. PMC 2728378. PMID 17406547. https://www.photophysics.com/circular-dichroism/chirascan-technology/chirality- and-cd/

CD experiments Lab: F040A Assistants: Hongmin & Abhi Prepare samples* and blank controls in suitable buffers. Learn to operate the instrument: Chirascan™-plus CD Spectrometer (Applied Photophysics). Measure blank and sample CD spectrometry. Measure melting temperature. Learn data analysis softwares: Pro-Data Viewer (CD), Globe3 (Tm), CDNN (secondary structure deconvolution). *Protein concentration, MW, amino acid numbers should be checked before the analysis.

Report CD results Your own sample: Compare your sample with others: CD curve table of secondary structure deconvolution result Tm result. Compare your sample with others: same constructs others Discussion: why are there any differences?

II. Isothermal titration calorimetry (ITC) Measure interactions between two or more molecules in solution (why does it happen?) protein ligand

Free energy change, G, is a measure of spontaneity of a process and of the usefull energy available from such a process. Forces driving the interaction: enthalpy change (H) and entropy change (S)

Δ G = -RT ln(KA) =Δ H − T Δ S (R = gas constant 1.9858775 cal K-1 mol-1, T = absolute temperature 273.15K = 0 °C)

ITC measurement First injection results in a larger deflection from the baseline. At the end, little or no deflection from baseline. Heat is given off during the reaction, therefore less power is required to compensate the temperature differences. Measures the heat produced or consumed by the interaction reaction. Titration to determine the endpoint (related to the interaction stoichiometry).

ITC data analysis Titration curve is fitted to a binding model to calculate the affinity (KD), stoichiometry (n) and the enthalpy of interaction (ΔH). The individual injection heats are calculated by integrating the raw data (power) from each injection over time. The fitted curve of a binding model is overlaid in red. Thermodynamic parameters n, KD, and ΔH are calculated.

ITC applications Non-covalent complexes: molecules interact through a combination of multiple weak interactions (hydrogen bonds, electrostatic interactions, van der Waals interactions, and hydrophobic interactions, etc.). Interaction between any type of molecules from ions and polymers to nanoparticles and biomolecules. Mostly used in protein-protein, protein-DNA, protein-membrane, or DNA-ligand binding. Provides valuable information at different levels: whether or not two given molecules interact; binding stoichiometry; binding affinity or strength of the complex formation; partition of the Gibbs energy of binding into enthalpy and entropy contributions.

ITC technical limitation Advantages Thermodynamic characterization (stoichiometry, association constant, and binding enthalpy) in a single experiment. No need for reporter labels (e.g. chromophores, fluorophores). Direct determination of the binding enthalpy. Interaction in solution (no need for reactant immobilization). Possibility of performing experiment with optically dense solutions or unusual systems (e.g. dispersions, intact organelles or cells). …

ITC technical limitation (continued) Disadvantages Signal is proportional to binding enthalpy, and non-covalent complexes may exhibit rather small binding enthalpies. Heat is a universal signal, and every process will contribute to the global measured heat, thus complicating the evaluation of the contribution due to binding. High sample amount: 300 µl sample (concentration ≥10 µM) and 60 µl ligand (≥10 times of sample concentration) are needed. Slow technique with a low throughput (0.25 – 2 h/assay), not suitable for HTS. Kinetically slow processes may be overlooked. Limited association constants 104 – 109 M-1. No automatic injection. …

ITC example Drug screening: binding of antibiotic Lividomycin to HIV-1 genomic RNA Dimerization Initiation Site (DIS)

Binding conditions optimized with ITC provide useful information for X-ray crystal structure of RNA-lividomycin complex

References https://www.malvernpanalytical.com/en/products/technology/microcalorimetry/isothermal-titration-calorimetry/ https://pubs.acs.org/doi/pdfplus/10.1021/ac00217a002

ITC experiments Lab: F040A Assistants: Hongmin & Abhi Prepare samples in HBS buffer. Learn to operate the instrument: MicroCal ITC200 (Malvern). Measure binding of NiCl2 to His-tagged proteins. Learn data analysis software: Origin

Report ITC results Raw titration curve Final analysis Calculated values: KD, N, ΔH, ΔS, ΔG Discussion: KD value, compared with SPR results Stoichiometry (N=1?) How to improve the data quality?

III. Surface Plasmon Resonance (SPR) Optical technology detecting refractive index changes at the surface of a metallic film. The response values of SPR are expressed in resonance units (RU) 1 RU = 0.0001°. ~1 pg/mm2 at the sensor surface.

SPR measurement Real-time detection of biomolecular interaction Quantitative studies on: (1) specificity; (2) affinity; (3) Kinetics; (4) Concentration

Proportion of complex and free interactants at equilibrium SPR data analysis Original binding data are fitted to mathematic models. Binding parameters are calculated from the model formula. Proportion of complex and free interactants at equilibrium Rate

SPR application Screening and detecting binding partners: drug discovery Kinetics and affinity measurements: biomolecule complex formation Concentration measurements: pharmaceutical and food industry Mapping binding sites: antibody specificity or epitope mapping studies Biosensor development Material science

Binding of indometacin to human serum albumin (HSA) SPR example Binding of indometacin to human serum albumin (HSA) Sensorgram suggests fast association and dissociation. Data fitting to models suggests two binding sites

SPR technical limitation Advantages Sensitive. Low sample amount needed. Provides both affinity and kinetics parameters. … Disadvantages Good experimental design is needed. Fitting experimental data to the binding model is not easy. Slow, difficult for high throughput analysis.

References www.sprpages.nl Handbook of Surface Plasmon Resonance • SPR Instrumentation / Richard B.M. Schasfoort and authors of instrument providers • Kinetics of biomolecular interactions / Peter Schuck • SPR pages. Getting a feeling for the curves / Arnoud Marquart • Surface Chemistry in SPR Technology / Erk T. Gedig • Treating raw data: Software for SPR applications Available at http://pubs.rsc.org/ ISBN 978-1-78262-730-2

SPR experiments Lab: F040A Assistants: Hongmin & Abhi Prepare samples in HBS-running buffer. Learn to operate the instrument: Biacore T200 (GE), or MP-SPR Navi 220A Naali. Measure binding of NiCl2 to His-tagged proteins. Data analysis. Options: try with less Ni2+ immobilized, more salt or surfactant in running buffers

Report SPR results together with ITC results Raw sensorgrams Analyzed data: affinity and kinetics fitting curves Calculated values: KD, ka, kd Discussion: KD value, from affinity analysis and from kd/ka, compared with ITC results How to improve the data quality? Are there any weak points in the experiment design?