Biomolecular interaction analysis (BIA) techniques

1. University of Tehran Microbial Technology and Products (MTP) Research Center Biomolecular Interaction Analysis (BIA) Techniques By: Naghmeh Poorinmohammad November…
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  • 1. University of Tehran Microbial Technology and Products (MTP) Research Center Biomolecular Interaction Analysis (BIA) Techniques By: Naghmeh Poorinmohammad November 2014
  • 2. • Introduction • BIA techniques 1. Biochemistry & biophysics methods 2. Molecular Methods 3. Computer-aided techniques 4. Novel creative approaches • Summary/Conclusion • References 2 Outline
  • 3. 3 Outline • Introduction • BIA techniques 1. Biochemistry & biophysics methods 2. Molecular Methods 3. Computer-aided techniques 4. Novel creative approaches • Summary/Conclusion • References
  • 4. Biomolecular Interaction Analysis (BIA) • Almost all biological processes can be described as a sequence of interaction events between macromolecules/small molecules. • The study of such interactions (BIA) is key for: 1. Understanding of biology at the molecular level 2. Drug discovery 3. Development of diagnostic tests • It is thus crucial to develop techniques that can: 1. Probe 2. characterize 3. Discriminate 4
  • 5. Outline • Introduction • BIA techniques: 1. Biochemistry and biophysics methods 2. Molecular Methods 3. Computer-aided techniques 4. Novel creative approaches • Summary/Conclusion • References 5
  • 6. 1. Biochemistry and biophysics methods Optical Fluorescence SPR MST ATR-FTIR Raman MS NMR Electrochemistry Equilibrium dialysis Chromatography ITC DSC Electrophoresis UV Non-optical CD Pull-down 6
  • 8. Fluorescence spectroscopy • Fluorescence originates from electronic transitions in molecules with conjugated bonds. • When a fluorophore, which is excited by a photon, returns from an excited singlet electronic state to the ground singlet electronic state, it may emit light in the form of fluorescence. • The observable fluorescence intensity, If , is a wavelength dependent quantity and is related to the quantum yield and optical density  quantitative treatment of fluorescence binding data. • In general, the optimal spectroscopic conditions should be selected to exploit the properties of the excitation and emission scans of the complex and its constituents. Mocz, Gabor, and Justin A. Ross. "Fluorescence Techniques in Analysis of Protein–Ligand Interactions." Protein-Ligand Interactions. Humana Press, 2013. 169-210. 8
  • 9. Fluorescence Resonance Energy Transfer (FRET) From: Broussard et al. 2013; nature protocols • Donor and acceptor molecules must be in close proximity (10–100 Å). • The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor. Song, Yang, et al. "Protein interaction affinity determination by quantitative FRET technology." Biotechnology and bioengineering 109.11 (2012): 2875-2883. 9 • The donor absorption and emission spectra should have a minimal overlap to reduce self-transfer.
  • 10. Fluorescence Resonance Energy Transfer (FRET) From: 10
  • 11. Fluorescence Resonance Energy Transfer (FRET) • Intensity ≈ [sample]; when OD< 0.05 • In greater OD values the relation is non-linear, thus complicated for calculations. Song, Yang, et al. "Protein interaction affinity determination by quantitative FRET technology." Biotechnology and bioengineering 109.11 (2012): 2875-2883. 11 • Binding free energy can be derived from: ΔG= RTlnKd
  • 12. Fluorescence Resonance Energy Transfer (FRET) Advantages Drawbacks Allows the study in living cells High background noise Short time of trial (<90s) Donor reabsorbs emission Quantitative Natural fluorescence of cells Sensitive Oxygen quenching effect Needs low concentration of sample External light source required : Here comes BRET 12
  • 13. Ultra Violet-visible (UV-Vis) spectroscopy • Absorption spectroscopy in the visible and ultraviolet spectral regions is a powerful technique by which ligand binding equilibria can be studied. Three different internal chromophores in Biomolecule The peptide bond (absorbs weakly 220 nm) Aromatic amino acids F, Y, W, H (230–300 nm) Many biological molecules show strong absorbance in the visible region of the spectrum as a result of the presence of metal ions and prosthetic groups with extended π-electron systems, such as chlorophyll, carotinoid, flavin, heme. Nienhaus, Karin, and G. Ulrich Nienhaus. "Probing heme protein-ligand interactions by UV/visible absorption spectroscopy." Protein-Ligand Interactions. Humana Press, 2005. 215-241. 13 These bands are sensitive to the surrounding polypeptide environment and reflect structural changes, oxidation states, and the binding of ligands.
  • 14. 15
  • 15. Ultra Violet-visible (UV-Vis) spectroscopy Advantages Drawbacks Simple (more than with fluorescence) Less sensitive than fluorescnece Inexpensive Less specific than fluorescence Quantitative Overlap bands 16
  • 16. Circular Dichroism (CD) Spectroscopy • CD is the difference in absorption of left and right circularly polarized light. • Proteins and DNA and many ligands are chiral. • Molecular interactions between chiral and achiral compounds can give rise to induced circular dichroism (ICD) of the achiral counterpart. • If it is chiral then its ICD is the difference between its own CD spectrum and the spectrum in the presence of the protein. Rodger, Alison, et al. "Circular dichroism spectroscopy for the study of protein-ligand interactions." Protein-Ligand Interactions. Humana Press, 2005. 343-363. 17
  • 17. Circular Dichroism (CD) Spectroscopy Daviter, Tina, Nikola Chmel, and Alison Rodger. "Circular and linear dichroism spectroscopy for the study of protein– ligand interactions." Protein-Ligand Interactions. Humana Press, 2013. 211-241. 18
  • 18. Circular Dichroism (CD) Spectroscopy Advantages Drawbacks Gives structural information Machines are more expensive than UV Sensitive Measures binding strength of the order of μM Relatively rapid (20 min) - Quantitative - No extensive sample preparation - 19
  • 19. Nuclear Magnetic Resonance (NMR) Spectroscopy • A physical phenomenon in which nuclei in a magnetic field absorb and re-emit electromagnetic radiation. • NMR detects ligand binding through changes in the resonant frequencies (chemical shifts) of NMR-active nuclei. • NMR spectroscopy detects and reveals protein ligand interactions with a large range of affinities (10 9–10-3 M). • Protein samples need to be isotopically enriched (15N and/or 13C). Mittermaier, Anthony, and Erick Meneses. "Analyzing Protein–Ligand Interactions by Dynamic NMR Spectroscopy." Protein-Ligand Interactions. Humana Press, 2013. 243-266. 20 • Larger molecules (>25 kDa), additional enrichment with 2H. • Isotopically labeled protein  over-express in bacteria grown in minimal media containing 15NH4Cl and/or 13C glucose as the sole sources of nitrogen and/or carbon.
  • 20. Nuclear Magnetic Resonance (NMR) Spectroscopy Advantage Drawback Very sensitive to weak interactions Needs concentrated isotopically lablled sample 50μM- 2mM) Reveals the portion of molecule involved in interaction Not suitable for >100 KDa Accurate kinetics even for short lifetime bounds (< 1ms) Needs high purity sample Assay in equilibrium solution Requires ligand-receptor buffer harmony Quantitative (large range of affinities) Strong magnetic fields needed for high quality  expensive - Long assay time 21
  • 21. Mass Spectroscopy • Using ESI-MS, it is possible to transfer weakly associated complexes from solution into the gas phase inside the mass spectrometer source. • ESI-MS not only provides a direct readout of binding stoichiometry but can also be used to determine dissociation constants ranging from nM to mM. • The number of ligands bound for a given protein–ligand system can be determined directly from the spectrum based on the mass difference between free protein and its ligated complexes. • In addition to exploiting the ‘x axis’ of the mass spectrum (that is, the mass-tocharge ratio, m/z), the ‘y axis’ of the mass spectrum (that is, abundance/intensity) provides important information about affinity and specificity. - Pacholarz, Kamila J., et al. "Mass spectrometry based tools to investigate protein–ligand interactions for drug discovery." Chemical Society Reviews41.11 (2012): 4335-4355. - Hofstadler, Steven A., and Kristin A. Sannes-Lowery. "Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes." Nature Reviews Drug Discovery 5.7 (2006): 585-595. 22
  • 22. Mass Spectroscopy Pacholarz, Kamila J., et al. "Mass spectrometry based tools to investigate protein–ligand interactions for drug discovery." Chemical Society Reviews41.11 (2012): 4335-4355. 23
  • 23. Surface-enhanced laser desorption/ionization (SELDI) • SELDI-TOF MS, combines two powerful techniques: chromatography and From: Tang et al. 2004; Mass spectrometry reviews mass spectrometry. • Different SELDI plates are available: CM10 (ion-exchange), H50 (like C6 to C12 reverse phase) 24
  • 24. Mass Spectroscopy Advantage Drawback Binding stochiometry and kinetics Does not discriminate between specific and non-specific binding Sensitive/specific lower sample consumption Does not provide information regarding the binding site of the ligand and structure changes High throughput capability (nano- ESI-MS) Instrumental appropriate tuning Label free - No stabilization needed - 25
  • 25. Fourier transform infrared (FTIR) spectroscopy • Interaction of infrared light with the vibrational modes of the molecules being interrogated. • High IR absorptivity of water  IR samples are very thin, the relative water . 26
  • 26. Attenuated Total Reflectance (ATR)-FTIR • ATR Molecule being studied may be in contact to water above, which is not sensed by the IR beam. • Its characteristics allow ligands to be detected only when they are bound to receptors closely linked to the sensor surface. From: Kazarian, Sergei G., and KL Andrew Chan. "ATR-FTIR spectroscopic imaging: recent advances and applications to biological systems." Analyst 138.7 (2013): 1940-1951. 27
  • 27. ATR-FTIR spectroscopy Advantage Drawback Rapid assay (10ms) Needs concentrated sample (2-10mM) label free Needs ATR correction software low-cost Stabilization process (in some cases) Low sample volume requirement (1/3 NMR) Quantitative determination Suitable for large/membrane proteins Low Sensitivity 28
  • 28. Surface Enhanced Raman Spectroscopy • It can be dramatically enhanced (102–1014) by adsorption of molecules on a surface of noble metal, transition metal, or semiconductor substrates (SERS). From: Siddhanta et al. 2012; Nanomaterials and Nanotechnology From: Siddhanta, Soumik, and Chandrabhas Narayana. "Surface Enhanced Raman Spectroscopy of Proteins: Implications in Drug Designing." Nanomaterials and Nanotechnology 2.1 (2012): 1-13. 29
  • 29. Raman IR Due to the scattering of light by the vibrating molecules. Result of absorption/reflectance of light by vibrating molecules. The vibration is Raman active if it causes a change in polarisability. The vibration is IR active if there is a change in dipole moment during the vibration. Many distinguishable peaks with high intensities Few distinguishable peaks with weak intensities (even in ATR-FTIR) Water can be used as a solvent. Water cannot be used due to its intense absorption (not for ATR). Sample preparation is not very elaborate sample can be almost in any state. Sample preparation is elaborate Gaseous samples can rarely be used. Cost of instrumentation is very high Comparatively inexpensive. 30
  • 30. Surface Plasmon Resonance (SPR) • SPR is the resonant oscillation of conduction electrons at the interface which will affect with polirized light. From: Stahelin, Robert V. "Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions." Molecular biology of the cell 24.7 (2013): 883-886. 31
  • 31. Surface Plasmon Resonance (SPR) Advantage Drawback Label free Tethering of molecules to surfaces may affect the binding constants measured Enables quantitative determination Any artifactual RI change other than from the interaction can also give signal Low sample volume requirement Stabilization process (in some cases) Real time assay Sensitive Cannot verify the stability of the complex formed 32
  • 32. Micro-scale thermophoresis (MST) • It measures the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size. • An infrared-laser is used to generate precise microscopic temperature gradients within thin glass capillaries that are filled with a sample in a buffer or bioliquid of choice. • Thermophoresis, is very sensitive to changes in size, charge, and solvation shell of a molecule and thus suited for bioanalytics. • The fluorescence of molecules is used to monitor the motion of molecules along these temperature gradients. The fluorescence can be either intrinsic (e.g. tryptophan) or of an attached dye or fluorescent protein (e.g. GFP). Jerabek-Willemsen, Moran, et al. "Molecular interaction studies using microscale thermophoresis." Assay and drug development technologies 9.4 (2011): 342-353. 33
  • 33. Micro-scale thermophoresis (MST) From: 34
  • 34. Micro-scale thermophoresis (MST) From: Jerabek-Willemsen et al. 2011; Assay and drug development technologies. 35
  • 35. Micro-scale thermophoresis (MST) 36
  • 36. Micro-scale thermophoresis (MST) 37
  • 37. Micro-scale thermophoresis (MST) 38
  • 38. Micro-scale thermophoresis (MST) Advantage Drawback Sample concentration (pM/nM) and small volume (< 4 μl) Buffer condition must be absolutely stable Quantitative (K: pM/nM to mM range and n) Conformational changes induced by IR-Laser heating may be problematic measures interactions with essentially no limitation on molecule size or molecular weight. - Immobilization free - Free in choosing buffer type - 39
  • 40. Isothermal Titration Calorimetry (ITC) • All chemical, physical and biologic processes are performed along with heat exchange criteria. • When a protein interacts with a ligand, heat is either released or absorbed. • ITC relies only on the detection of a heat effect upon binding  not relies on the presence of chromophores or fluorophores. • Can be used to measure the binding constant, the enthalpy of binding, and the stoichiometry. • Modern instruments, like the MicroCal and Calorimetry Sciences Corporation ITCs, make it possible to measure heat effects as small as 0.4 μJ (0.1 μcal) allowing the determination of binding constants, K’s, as large as 108 to 109 M–1. Lewis, Edwin A., and Kenneth P. Murphy. "Isothermal titration calorimetry."Protein-Ligand Interactions. Humana Press, 2005. 1-15. 41
  • 41. Isothermal Titration Calorimetry (ITC) • The linear parameters ΔH and n will be better determined than the nonlinear parameter K. • The best results will be obtained at 10/K <[M] <100/K, and [L] 20 to 50 × [M], subject to solubility and heat signal considerations. • Titrant and titrate concentrations must be accurately known. • (Nonintegral values for n are often the result of concentration errors. Errors in titrate concentration contribute directly to a similar systematic error in n. Errors in titrant concentration or titrant delivery contribute directly to errors in K and ΔH.) Lewis, Edwin A., and Kenneth P. Murphy. "Isothermal titration calorimetry."Protein-Ligand Interactions. Humana Press, 2005. 1-15. 42
  • 42. Isothermal Titration Calorimetry (ITC) 43
  • 43. Isothermal Titration Calorimetry (ITC) 43
  • 44. Isothermal Titration Calorimetry (ITC) 43
  • 45. Isothermal Titration Calorimetry (ITC) 43
  • 46. Isothermal Titration Calorimetry (ITC) 43
  • 47. Isothermal Titration Calorimetry (ITC) 43
  • 48. Isothermal Titration Calorimetry (ITC) 43
  • 49. Isothermal Titration Calorimetry (ITC) Lewis, Edwin A., and Kenneth P. MFurropmhy:. h"tItspo:/t/hwerwmwa.le ntidtroactiyotons cisa.loorrgim/teectrhyn."iqPsr/oITteCin.h-Ltmigland Interactions. Humana Press, 2005. 1-15. 44
  • 50. Isothermal Titration Calorimetry (ITC) Advantage Drawback Label free Large sample volumes required Enables quantitative determination (K and n) High ligand concentrations Can be done on solutions that are either homogeneous or heterogenous Presence of impurities or inactive protein will have a direct impact on the stoichiometry Universal - 45
  • 51. Differential Scanning Calorimetry (DSC) • Measures heat capacity in a range of temperatures. • If a ligand binds preferentially to the native state of the protein, the temperature at which the protein-ligand complex denatures will be higher compared to the temperature at which the free protein unfolds. • Since the degree of stabilization or destabilization of the native protein depends on the magnitude of the binding energy, comparison of the stability of the complex with the stability of the ligand-free protein allows the binding energy to be estimated. • DSC thus provides a direct measure of whether ligand binding to a protein is stabilizing or destabilizing, and so can complement studies of binding equilibria obtained by isothermal titration calorimetry (ITC). Chiu, Michael H., and Elmar J. Prenner. "Differential scanning calorimetry: an invaluable tool for a detailed thermodynamic characterization of macromolecules and their interactions." Journal of Pharmacy And Bioallied Sciences 3.1 (2011): 39. 46
  • 52. Differential Scanning Calorimetry (DSC) 47
  • 53. Differential Scanning Calorimetry (DSC) Advantage Drawback Label free Sensitivity depends on many parameters Quantitative (relatively) useful in characterizing very tight binding interactions which equilibrate very slowly (mins to hrs) Gives information on the nature of binding event - 48
  • 54. Equilibrium Dialysis • The molecular weight cut off (MWCO) is chosen such that it will retain the receptor component. • A known concentration and volume of ligand is placed into one of the chambers. The ligand is small enough to pass freely through the membrane. • A known concentration of receptor is then placed in the remaining chamber in an equivalent volume to that placed in the first chamber. • A complete binding curve is generated by measuring Y at different ligand concentrations. • The relationship between binding and ligand concentration is then used to determine the number of binding sites, the ligand affinity, kd. Because this kind of experimental data used to be analyzed with (Scatchard plots) Hatakeyama, Tomomitsu. "Equilibrium Dialysis Using Chromophoric Sugar Derivatives." Lectins. Springer New York, 2014. 165-171. 49
  • 55. Equilibrium Dialysis 50
  • 56. Equilibrium Dialysis Advantage Drawback Truely label-free Not very rapid Quantitative Large sample volumes Immobilization free - 51
  • 57. Affinity Capillary Electrophoresis (ACE) • The technique uses the resolving power of CE to distinguish between free and bound forms of a receptor as a function of the concentration of free ligand. • ACE experiments are most commonly performed in fused silica capillaries by injecting a receptor and neutral marker with increasing concentrations of ligand in the separation buffer. • By studying the mobility change of a certain molecule when it int
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