A Biophysical Toolkit for Molecular Interactions

Abstract

Intermolecular interactions inside and around the cell are key to maintain basic cell homeostasis and to sense and react to external stimuli. Moreover, the action of therapeutics and other xenobiotics occurs through physical interactions of these agents with biomolecules present in the cell or tissue, thereby interfering with the ‘native’ interactions of the biomolecules. Biophysical characterisation of molecular interactions is a bottom‐up reductionist approach that implies measuring the physicochemical properties of the interacting species (two biomacromolecules or a biomacromolecule and a small‐molecule ligand) in order to quantify the binding strength (thermodynamics) and association and dissociation speed (kinetics) between them. A large variety of macroscopic experimental techniques are available and constitute a biophysical toolkit to study molecular interactions in vitro. Among others, isothermal titration calorimetry, surface plasmon resonance, analytical ultracentrifugation, nuclear magnetic resonance, microscale thermophoresis, thermal shift and fluorescence‐based methods are most commonly used.

Key Concepts

  • The strength (affinity) and speed (kinetics) of interactions between biomolecules can be quantified using biophysical experimental methods.
  • Isothermal titration calorimetry (ITC), fluorescence and nuclear magnetic resonance (NMR) spectroscopies, thermal shift assay (TSA), analytical ultracentrifugation (AUC), microscale thermophoresis (MST) and surface plasmon resonance (SPR) are arguably the most used and versatile techniques to study binding in vitro and constitute a biophysical toolkit.
  • Equilibrium affinity constants can be robustly obtained by titration using ITC, fluorescence, NMR, MST, AUC and SPR.
  • Kinetic information can be obtained from time‐resolved fluorescence spectroscopy and SPR methods. The recently developed kinITC method allows obtaining kinetic information from ITC data.
  • Biophysical techniques can tackle the study of a wide range of systems and great progress has been made in the characterisation of interactions taking place at the cell membrane.
  • Basically, all biophysical methods can be automatised to screen for small‐molecule ligands of proteins and other macromolecules, providing starting points for chemical biology and drug discovery.

Keywords: biomolecular interactions; binding affinity; binding kinetics; isothermal titration calorimetry; surface plasmon resonance; analytical ultracentrifugation; nuclear magnetic resonance spectroscopy; microscale thermophoresis; thermal shift assay; fluorescence spectroscopy

Figure 1. ITC study of the human aspartate transcarbamoylase (ATCase) homotrimeric domain of the CAD protein, binding to its CP (carbamoyl phosphate) substrate and the PALA (N‐phosphonacetyl‐l‐aspartate) inhibitor. (a) CP binds exothermically and (b) PALA binds endothermically. Upper panels show the thermograms and lower plots show the binding isotherms (normalised heat per injection as a function of the molar ratio ligand/protein). Nonlinear fitting analysis was performed using three‐site noncooperative (for CP) and cooperative (for PALA) binding models, yielding the indicated intrinsic site‐specific dissociation constants. The substrate CP binds with the same affinity to three independent binding sites while only two of the three active sites show high affinity for the inhibitor PALA (negative cooperativity). Adapted from Ruiz‐Ramos et al. (2016) © Elsevier.
Figure 2. Fluorescence methods to characterise binding affinity and kinetics. (a, b) Titrations of mant‐GTP (guanosine triphosphate) probe (200 nM) with FtsZ protein from Bacillus subtilis using fluorescence intensity and anisotropy, respectively. Lines in each case correspond to the best fit to the data for a 1:1 binding. (c) Chemical structures of small ligands tested in the displacement assay. (d) Displacement curves of mant‐GTP (500 nM) from FtsZ (300 nM binding sites) with unmodified GTP and with these molecules, measured by fluorescence anisotropy. Compounds were checked not to interact with the mant‐GTP probe. The solid lines are best fits to the data according to a single site competition model. (e) Kinetics of binding of mant‐GDP (guanosine diphosphate) (1 μM) to Apo‐FtsZ (10 μM) fitted to a single exponential; the empty symbols are a control measurement of mant‐GDP mixed with buffer. (f) Kinetics of dissociation of mant‐GDP from FtsZ. The mant‐GDP‐FtsZ complex was formed by adding 2 μM Apo‐FtsZ to 1 μM mant‐GDP and at time 0, the solution was mixed 1:1 with 400 μM GTP; the grey line is a control of mant‐GDP alone. Data were fitted to a single exponential. (d–f) Modified from Ruiz‐Avila et al. (2013) and from Huecas et al. (2007) © American Chemical Society.
Figure 3. Differential scanning fluorimetry. (a) Fluorescence intensity versus temperature for protein thermal unfolding in the presence of the fluorescent dye SYPRO orange (represented as an aromatic ring). The fluorescence of the dye (excited by light of 492 nm and represented by green arrows) is quenched in the aqueous buffer where the protein is folded. As the temperature increases, the protein unfolds and its hydrophobic parts (white areas) become solvent exposed, resulting in a strong fluorescence emission at 610 nm (represented by red arrows) by the dye molecules bound to them. Tm is calculated by fitting the experimental data points (circles) to simple equations such as the indicated Boltzmann equation (LL and UL represent, respectively, the lower and upper limits of the dye‐emitted fluorescence intensity and a denotes the slope of the curve within Tm). Adapted from Niesen et al. () © Nature Publishing Group. (b) Stabilisation of His‐PA1 and His‐PTIP proteins upon complex formation measured by nanoDSF. The His‐PA1/His‐PTIP complex has a Kd of 7.1 ± 2.4 nM at 25 °C as measured by ITC. His‐PA1 binding stabilises His‐PTIP by more than 15 °C. The fluorescence intensity ratio at 350 and 330 nm for the individual proteins (note that no thermal transition is observed for isolated His‐PA1) and their complex are fit to a polynomial function to extract the Tm values (upper panel). The lower panel represents the first derivative of the polynomial function and its maximum corresponds to the Tm. Two replicates per sample are overlaid. Adapted from Starnes et al. (2016) © Cold Spring Harbor Laboratory Press.
Figure 4. Sedimentation velocity boundaries collected at 42 000 rpm and 20 °C corresponding to (a) protein p6 dimer (22 kDa), (b) DNA (deoxyribonucleic acid, fragment L 256 base pairs duplex) and (c) the p6–DNA mixture. Absorbance was measured at the selected wavelength (in most of the cases at 255 or 275 nm). (d) The resulting sedimentation coefficient distributions [c(s)] obtained after boundary analysis of protein p6 (dotted line), DNA (dashed line) and p6–DNA mixture (solid line). (e) Sedimentation equilibrium gradients taken at 5000 rpm and 20 °C of protein p6 (black circles), DNA (triangles) and p6–DNA mixture (white circles), equilibrated in 0.05 M NaCl buffer. Continuous lines represent the best‐fit single‐species model that accounts for the experimental data as described in the text. The lower panel shows the best‐fit residuals distribution. (f) Binding isotherms of protein p6 to DNA at low‐salt (black triangles) and high‐salt (white triangles) buffers measured by sedimentation equilibrium. Protein p6 dimer binds noncooperatively to 11 dimer sites within the DNA (the maximal binding capacity is 22 mol of p6 monomer per mole of DNA). The x‐axis corresponds to free protein concentration. Error bars represent ±2SD. Adapted from Alcorlo et al. (2009) © Elsevier.
Figure 5. Microscale thermophoresis determination of protein–peptide binding affinity. (a) Fluorescence time traces for the spindle checkpoint protein complex Bub1(1‐533)‐Bub3 interacting with an MDITp repeat (or pMELT12 peptide), one of the multiple phosphorylated MELT motifs of the outer kinetochore or KNL1 protein, as recorded with a Monolith NT.115 instrument. The concentration of the fluorescently labelled Bub1(1‐533)‐Bub3 (cysteine red labelled) was kept constant at 12.5 nM, and the concentration of the pMELT12 peptide varied from 2.44 nM to 20 μM. MST (microscale thermophoresis) traces at increasing ligand concentrations are colour coded from red to blue. The subsequent processes that affect the recorded fluorescence are labelled from I to V and described in the main text. (b) Binding titrations of two single phosphorylated MELT repeats to fluorescently labelled Bub1(1‐533)/Bub3. pMELT12 shows low micromolar affinity, Kd = 579 ± 133 nM (red circles), while pMELT18 (blue triangles) does not show binding. Lines represent best fits of the data points using the law of mass action (for pMELT12) or the average of experimental data points (for pMELT18). The error bars represent the SD of each data point calculated from three independent thermophoresis experiments (for pMELT12) or the SD of the data points (for pMELT18). Adapted from Zhang et al. (2016) © Nature Publishing Group.
Figure 6. Affinity determination of a small molecule binding telomeric DNA (deoxyribonucleic acid) quadruplexes using receptor‐observed NMR (nuclear magnetic resonance). (a) Representation of the chemical shift changes in the imino region of the 1H NMR spectrum of a telomeric DNA guanine quadruplex model (dTERRA2, a bimolecular symmetric quadruplex, at 33 μM) upon titration with a small‐molecule compound (MW = 322 g mol−1). A–F refer to the six imino signals detected in the 1H NMR spectrum of dTERRA2 and their positions are indicated in the free DNA (lower spectrum) and in the presence of 370 μM compound (upper spectrum). (b) Best nonlinear fits for determination of dissociation constants. Independent fit of three most affected imino signals of dTERRA2 upon compound titration yield Kd values of 130 ± 13 μM (A), 121 ± 20 μM (B) and 111 ± 15 μM (F). Adapted from Garavís et al. (2014) © American Chemical Society.
Figure 7. Binding analysis of protein–DNA interactions using SPR (surface plasmon resonance). (a) SPR sensorgrams (thick traces) of a DNA‐binding protein (BUD) injected at increasing concentrations (0.4, 0.8, 1.6 and 3.2 nM) over its biotin/streptavidin‐immobilised target DNA and determination of Kd from the ratio of kinetic rate constants, obtained from best‐fit (thin lines) kinetic analysis. (b) A different DNA‐binding protein (TALE) binds BUD DNA target with 5000‐fold weaker affinity (Kd = 1 μM, as obtained from best fit of the concentration dependence of steady‐state responses at different concentrations, insert). The BUDs (BUrrH Domains) are highly specific DNA‐binding proteins with a simple amino acid to base code and thus promising tools for genome engineering applications. Adapted from Stella et al. (2014) © International Union of Crystallography.
close

References

Alcorlo M, Jimenez M, Ortega A, et al. (2009) Analytical ultracentrifugation studies of phage phi29 protein p6 binding to DNA. Journal of Molecular Biology 385 (5): 1616–1629.

Alexander CG, Wanner R, Johnson CM, et al. (2014) Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings. Biochimica et Biophysica Acta‐Proteins and Proteomics 1844 (12): 2241–2250.

Alfonso C, del Castillo U, Martín I, Muga A and Rivas G (2015) Sedimentation equilibrium analysis of ClpB self‐association in diluted and crowded solutions. Methods in Enzymology 562: 135–160.

Barshop BA, Wrenn RF and Frieden C (1983) Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM‐a flexible, portable system. Analytical Biochemistry 130 (1): 134–145.

Boudker O and Oh S (2015) Isothermal titration calorimetry of ion‐coupled membrane transporters. Methods 76: 171–182.

Buey RM, Calvo E, Barasoain I, et al. (2007) Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites. Nature Chemical Biology 3 (2): 117–125.

Burnouf D, Ennifar E, Guedich S, et al. (2012) kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. Journal of the American Chemical Society 134 (1): 559–565.

Campos‐Olivas R (2011) NMR screening and hit validation in fragment based drug discovery. Current Topics in Medicinal Chemistry 11 (1): 43–67.

Clore GM and Iwahara J (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low‐population states of biological macromolecules and their complexes. Chemical Reviews 109 (9): 4108–4139.

Cole JL, Lary JW, Moody TP and Laue TM (2008) Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium. Methods in Cell Biology 84: 143–179.

Dalvit C (2008) Theoretical analysis of the competition ligand‐based NMR experiments and selected applications to fragment screening and binding constant measurements. Concepts in Magnetic Resonance Part A 32A (5): 341–372.

Dam J, Velikovsky CA, Mariuzza R, Urbanke C and Schuck P (2005) Sedimentation velocity analysis of protein‐protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s). Biophysical Journal 89 (1): 619–634.

Duhr S and Braun D (2006) Why molecules move along a temperature gradient. Proceedings of the National Academy of Sciences of the United States of America 103 (52): 19678–19682.

Fielding L (2007) NMR methods for the determination of protein‐ligand dissociation constants. Progress in Nuclear Magnetic Resonance Spectroscopy 51 (4): 219–242.

Freire E (2009) A thermodynamic approach to the affinity optimization of drug candidates. Chemical Biology & Drug Design 74 (5): 468–472.

Freyer MW and Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods in Cell Biology 84: 79–113.

Garavís M, López‐Méndez B, Somoza A, et al. (2014) Discovery of selective ligands for telomeric RNA G‐quadruplexes (TERRA) through 19F‐NMR based fragment screening. ACS Chemical Biology 9 (7): 1559–1566.

Helmerhorst E, Chandler DJ, Nussio M and Mamotte CD (2012) Real‐time and label‐free bio‐sensing of molecular interactions by surface plasmon resonance: a laboratory medicine perspective. Clinical Biochemist Reviews 33 (4): 161–173.

Huecas S, Schaffner‐Barbero C, Garcia W, et al. (2007) The interactions of cell division protein FtsZ with guanine nucleotides. Journal of Biological Chemistry 282 (52): 37515–37528.

Jerabek‐Willemsen M, Wienken CJ, Braun D, Baaske P and Duhr S (2011) Molecular interaction studies using microscale thermophoresis. Assay and Drug Development Technologies 9 (4): 342–353.

Kean J, Cleverley RM, O'Ryan L, et al. (2008) Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor‐based stability assay. Molecular Membrane Biology 25 (8): 653–663.

Kranz JK and Schalk‐Hihi C (2011) Protein thermal shifts to identify low molecular weight fragments. Methods in Enzymology 493: 277–298.

Le Maire M, Arnou B, Olesen C, et al. (2008) Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+‐ATPase as an example. Nature Protocols 3 (11): 1782–1795.

Le Roy A, Wang K, Schaack B, et al. (2015) AUC and small‐angle scattering for membrane proteins. Methods in Enzymology 562: 257–286.

Mizuguchi T, Uchimura H, Kataoka H, et al. (2012) Intact‐cell‐based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor. Analytical Biochemistry 420 (2): 185–187.

Monterroso B, Alfonso C, Zorrilla S and Rivas G (2013) Combined analytical ultracentrifugation, light scattering and fluorescence spectroscopy studies on the functional associations of the bacterial division FtsZ protein. Methods 59 (3): 349–362.

Nguyen HH, Park J, Kang S and Kim M (2015) Surface plasmon resonance: a versatile technique for biosensor applications. Sensors (Basel) 15 (5): 10481–10510.

Niesen FH, Berglund H and Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2 (9): 2212–2221.

Pantoliano MW, Petrella EC, Kwasnoski JD, et al. (2001) High‐density miniaturized thermal shift assays as a general strategy for drug discovery. Journal of Biomolecular Screening 6 (6): 429–440.

Patching SG (2014) Surface plasmon resonance spectroscopy for characterisation of membrane protein–ligand interactions and its potential for drug discovery. Biochimica et Biophysica Acta‐Biomembranes 1838 (1 Pt A): 43–55.

Pellecchia M, Bertini I, Cowburn D, et al. (2008) Perspectives on NMR in drug discovery: a technique comes of age. Nature Reviews in Drug Discovery 7 (9): 738–745.

Rajarathnam K and Rösgen J (2014) Isothermal titration calorimetry of membrane proteins — Progress and challenges. Biochimica et Biophysica Acta‐Biomembranes 1838 (1 Pt A): 69–77.

Raynal B, Lenormand P, Baron B, Hoos S and England P (2014) Quality assessment and optimization of purified protein samples: why and how? Microbial Cell Factories 13: 180.

Renaud JP, Chung C, Danielson UH, et al. (2016) Biophysics in drug discovery: impact, challenges and opportunities. Nature Reviews Drug Discovery 15 (10): 679–698.

Ruiz‐Avila LB, Huecas S, Artola M, et al. (2013) Synthetic inhibitors of bacterial cell division targeting the GTP‐binding site of FtsZ. ACS Chemical Biology 8 (9): 2072–2083.

Ruiz‐Ramos A, Velázquez‐Campoy A, García‐Grande A, Moreno‐Morcillo M and Ramón‐Maiques S (2016) Structure and functional characterization of human aspartate transcarbamoylase, the target of the anti‐tumoral drug PALA. Structure 24 (7): 1081–1094.

Salamon Z, Wang Y, Soulages JL, Brown MF and Tollin G (1996) Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films. Biophysical Journal 71 (1): 283–294.

Schaffner‐Barbero C, Gil‐Redondo R, Ruiz‐Avila LB, et al. (2010) Insights into nucleotide recognition by cell division protein FtsZ from a mant‐GTP competition assay and molecular dynamics. Biochemistry 49 (49): 10458–10472.

Schreiber G, Haran G and Zhou H‐X (2009) Fundamental aspects of protein–protein association kinetics. Chemical Reviews 109 (3): 839–860.

Schuck P (2013) Analytical ultracentrifugation as a tool for studying protein interactions. Biophysical Reviews 5 (2): 159–171.

Seidel SA, Wienken CJ, Geissler S, et al. (2012) Label‐free microscale thermophoresis discriminates sites and affinity of protein‐ligand binding. Angewandte Chemie International Edition 51 (42): 10656–10659.

Seidel SA, Dijkman PM, Lea WA, et al. (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59 (3): 301–315.

Shepherd CA, Hopkins AL and Navratilova I (2014) Fragment screening by SPR and advanced application to GPCRs. Progress in Biophysics and Molecular Biology 116 (2‐3): 113–123.

Smith AE, Zhang Z, Pielak GJ and Li C (2015) NMR studies of protein folding and binding in cells and cell‐like environments. Current Opinion in Structural Biology 30: 7–16.

Starnes LM, Su D, Pikkupeura LM, et al. (2016) A PTIP‐PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex. Genes & Development 30 (2): 149–163.

Stella S, Molina R, López‐Méndez B, et al. (2014) BuD, a helix‐loop‐helix DNA‐binding domain for genome modification. Acta Crystallographica. Section D, Biological Crystallography 70 (Pt 7): 2042–2052.

Turnbull WB and Daranas AH (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry? Journal of the American Chemical Society 125 (48): 14859–14866.

Van de Linde S, Heilemann M and Sauer M (2012) Live‐cell super‐resolution imaging with synthetic fluorophores. Annual Review of Physical Chemistry 63: 519–540.

Zhang G, López‐Méndez B, Sedgwick GG and Nilsson J (2016) Two functionally distinct kinetochore pools of BubR1 ensure accurate chromosome segregation. Nature Communications 7: 12256.

Zhao H, Fu Y, Glasser C, et al. (2016) Monochromatic multicomponent fluorescence sedimentation velocity for the study of high‐affinity protein interactions. eLife 5: e17812.

Further Reading

Aristotelous T, Ahn S, Shukla AK, et al. (2013) Discovery of β2 adrenergic receptor ligands using biosensor fragment screening of tagged wild‐type receptor. ACS Medicinal Chemistry Letters 4 (10): 1005–1010.

Cavanagh J, Fairbrother WJ, Palmer AG III and Skelton NJ (1996) Protein NMR Spectroscopy, Principles and Practice. New York, NY: Academic Press.

Cole JL (ed) (2015) Analytical Ultracentrifugation, vol. 562, pp. 2–567. Methods in Enzymology. London, UK: Academic Press.

Falconer RJ (2016) Applications of isothermal titration calorimetry ‐ the research and technical developments from 2011 to 2015. Journal of Molecular Recognition 29 (10): 504–515.

Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, 3rd edn. New York, NY: Springer.

Ma J, Metrick M, Ghirlando R, Zhao H and Schuck P (2015) Variable‐field analytical ultracentrifugation: I. Time‐optimized sedimentation equilibrium. Biophysical Journal 109 (4): 827–837.

Ma J, Zhao H, Sandmaier J, Liddle JA and Schuck P (2016) Variable field analytical ultracentrifugation: II. Gravitational sweep sedimentation velocity. Biophysical Journal 110 (1): 103–112.

Navratilova I, Besnard J and Hopkins AL (2011) Screening for GPCR ligands using surface plasmon resonance. ACS Medicinal Chemistry Letters 2 (7): 549–554.

Schasfoort RBM and Tudos AJ (eds) (2008) Handbook of Surface Plasmon Resonance. Cambridge, UK: Royal Society of Chemistry.

Velázquez Campoy A, Leavitt SA and Freire E (2004) Characterization of protein‐protein interactions by isothermal titration calorimetry. Methods in Molecular Biology 261: 35–54.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Santiveri, Clara M, López‐Méndez, Blanca, Huecas, Sonia, Alfonso, Carlos, Luque‐Ortega, Juan R, and Campos‐Olivas, Ramón(Jan 2017) A Biophysical Toolkit for Molecular Interactions. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027015]