1. 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

2. 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

3. 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.

4. CD spectrophotometer

5. CD measurements

6. Protein secondary structures

7. 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.

8. CD examples
wild type protein
mutant protein

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

10. References
Protein Circular Dichroism Data Bank:
Whitmore L, Wallace BA (2008). "Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases". Biopolymers 89 (5): 392–400. doi: /bip PMID
Greenfield NJ (2006). "Using circular dichroism spectra to estimate protein secondary structure". Nature protocols 1 (6): 2876–90. doi: /nprot PMC PMID
and-cd/

11. 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.

12. 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?

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

14. 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)

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

17. 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).

18. 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.

19. 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.

20. 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). …

21. 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. …

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

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

24. References

25. 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

26. 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?

27. 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 = °. ~1 pg/mm2 at the sensor surface.

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

29. 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

32. 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

33. 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

34. 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.

35. 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 ISBN

36. 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

37. 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?