Thought Leaders

Conformation Activity Relationships: Why Do Molecules Change Shape?

Dr. Gerry Ronan, CEO, Farfield Group Ltd.
Corresponding author: [email protected]


Biochemistry is characterised by weak, non covalent, bonds between large biopolymers which are continuously made and broken at varying reaction strengths. The interaction forces, typified by van der Waal's type forces can vary by the interaction distance to the power of 6 and therefore the ability of one molecule to 'fit' or conform to the shape of a fold or pocket on another drives the reaction.

Conversely, a binding partner can distort the conformation of a biomolecule (e.g. a protein) to enable or disable a potential biochemical activity thereby regulating the reaction. Indeed, this is the basic premise behind pharmaceutical intervention where small molecules are designed for their ability to selectively interact and distort the conformation of a target protein implicated in a disease mechanism.

Dual-Polarization Interferometry (DPI) measures the conformation of a protein by measuring its diameter (or size) and the density (i.e., its mass per unit volume or how tightly folded it is) by coupling the protein to a glass slide and probing using non-diffractive optics. The method resolves protein conformation to subatomic dimensions (well below 0.1 Å) in real time and has a growing acceptance among researchers in the field of protein characterization, an essential discipline in the science of proteomics.

In Farfield, our work over the past decade has revolved around the direct measurement of the shape or conformation of biomolecules and how this changes as the biomolecules function. This ability to monitor the Conformation Activity Relationship (CAR) is manifested in a benchtop analytical tool known as a Dual Polarisation Interferometer1 which measures the molecular size and fold density (and therefore mass) of interacting biomolecules captured on a glass slide. The technique has picometer resolution, is real time and label free and already has a growing userbase across 19 countries worldwide.

The Importance of Conformation Activity Relationships (CAR)

For example in pharmaceutical design, a conventional drug screening program will selects candidates from a library of many thousand if not millions on the basis of their ability to stick selectively to the protein target (as implicated in the disease mechanism), referred to as the affinity of the interaction. A high affinity interaction can occur at very low concentrations of the candidate and therefore the candidate is less likely to induce side effects elsewhere.However, high affinity gives no assurance that the candidate is conforming the biomolecule correctly or at all. Wouldn't it be better to have a lower affinity molecule, inducing the correct conformation for the desired activity which could then be optimised (by molecular engineering) to enhance its affinity?

A simple example is shown below where a target protein (prion protein as implicated in nvCJD) is challenged by different concentrations of a number of metal ions. From the mass of metal ion associated at each concentration the affinity (and stoichiometry) can be measured. However from the size and density profiles one can see immediately that cobalt does not distort the molecule while zinc, which has a similar affinity, does.

Furthermore, the conformational change evident in the case of copper (a compaction shown as a decrease in the size of the protein and an increase in its density) does not fully reverse (i.e. it has flipped into a different stable conformation or isoform) whereas the conformational change with zinc reverses fully. Three different Conformation Activity Relationships from three interactions with similar affinity.

Figure 1. Examples of different Conformation Activity Relationships for similar affinity binding interactions of metal ions binding to prion protein (PrP). Shown are the mass of association and dissociation of different metal ions at different concentrations (from which the interaction affinity can be calculated) and the corresponding size and density profiles (from which the conformational changes can be measured). The maximum conformational change (Cu) is 0.04nm and each challenge was a 5 minute injection. (Data courtesy of Gifu Univ., Japan)

Conformational Changes in Thin Films

Soft matter (e.g. polymers) is also subject to similar distortions and often it is this malleable nature that is the defining feature at the nanoscale. Dual Polarisation Interferometry (DPI) is also capable of characterising these changes in polymers, measuring both the film thickness and refractive index and agreement with other analytical techniques such as neutron reflection and ellipsometry2 is excellent as shown below in Figure 2. Unlike ellipsometry, however, DPI will determine the thickness and RI independently of each other at arbitrarily thin layers and unlike neutron data, it will do this in real time in a benchtop format with both experimental and control channels.

Figure 2. Comparison of DPI and ellipsometery measurements of a polyelectrolyte multilayer construct. At thick layers agreement for thickness (d) and refractive index (n) is excellent. At reduced thicknesses ellipsometry requires the knowledge of the RI (or thickness) in order to calculate the thickness (or RI) while DPI can measure arbitrarily thin layers revealing the oscillation in density associated with alternate positive and negatively charged layers depositing. (Data courtesy of YKI, Stockholm)

Many other examples of such self assembled constructs have been studied for example DNA multilayers3, chitosan/heparin4, and polyelectrolytes5,6.

Of course there are many types of conformational reorganisation of interest which do not involve a binding event. Polymer swelling due to changing pH is one such process which can quickly and easily be characterised again at a resolution normally associated with 'big physics'. A simple example is shown in Figure 3 where a surface captured thin film of poly(allylamine) is studied across a range of pH's. At low pH, the protonation of the polymer causes the layer to swell while at high pH the layer contracts and the density increases.

Figure 3. Polymer swelling due to protonation as measured by Dual Polarisation Interferometry

These measurements can also be extended to biopolymers where transitions from one isoform to another and also isoform stability can be characterised in a matrix of different temperatures, pH's, ionic strengths, solvents or other environmental or refolding conditions.

The Future of Dual Polarisation Interferometry

Since its introduction in 20037, Dual Polarisation Interferometry has been adopted by a wide range of researchers around the world in both the life and physical sciences. Its ability to measure and characterise molecular conformation at sub atomic resolution has created fundamentally new opportunities for nano and bio science research. The latest generation of instrumentation, the 4D Bio WorkStation is capable of an extended temperature to 65°C which allows the measurement of protein melt and other molecular phase transitions.

By characterising the kinetics and affinity of interactions at different temperatures it is also possible to quantify the free energy, enthalpy and entropy of not only binding but also conformational changes8. This allows for the first time the direct measurement of affinity, kinetics, thermodynamics and conformational change of binding or simply refolding in a single experiment.

Figure 4. A Dual Polarisation Interferometer benchtop instrument with automated sample introduction.

In the future higher throughput and smaller sample volumes will be required for screening applications but there are many other levels of spectroscopic information that can also be extracted.from the 4D Bio WorkStation. Measurement of very early stage protein crystal nucleation9 has already been demonstrated using optical loss whilst birefringence is now being used to measure order and disorder in lipid bilayers10 to characterise protein lipid interactions11. Our vision for the next decade is to enhance the fidelity of characterisation in these dimensions as well to truly illuminate the molecular world!


1. Swann M.J., Freeman N.J, Cross G. Dual Polarization Interferometry: A Real-Time Optical Technique for Measuring (Bio) Molecular Orientation, Structure and Function at the Solid/Liquid Interface. In: Handbook of Biosensors and Biochips, 2 Volume Set (2007). Eds: R. S. Marks, C. R. Lowe, D. C. Cullen, H. H. Weetall, I. Karube. Wiley, ISBN: 978-0-470-01905-4, Vol1, part 4, ch33, pp549-568.
2. Halthur T., Claessen P., Elofsson U., Immobilization of Enamel Matrix Derivate Protein onto Polypeptide Multilayers, Comparative in Situ Measurements Using Ellipsometry, Quartz Crystal Microbalance with Dissipation, and Dual-Polarization Interferometry. Langmuir (2006) 22(26)11065-71.
3. Lee L., Johnston A.P., Caruso F., Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length. Biomacromolecules (2008 )Nov. 9(11):3070-8. Epub 2008 Oct 1.
4. Lundin M., Blomberg E., Tilton R. D., Polymer Dynamics in Layer-by-Layer Assemblies of Chitosan and Heparin, Langmuir, Articles ASAP, Publication Date (Web): November 18, (2009) (Article) DOI: 10.1021/la902968h.
5. Aulin C., Varga I., Claesson P.M., Wågberg L., Lindström T., Buildup of polyelectrolyte multilayers of polyethyleneimine and microfibrillated cellulose studied by in situ dual-polarization interferometry and quartz crystal microbalance with dissipation. Langmuir, (2008) Mar 18; 24(6):2509-18. Epub (2008) Feb 16.
6. Lane T.J., FletcherW. R., Gormally, M. V., Johal M.S., Dual-Beam Polarization Interferometry Resolves Mechanistic Aspects of Polyelectrolyte Adsorption. Langmuir, (2008) ASAP Article, Web Release Date: September 10, (2008).
7. Swann M.J., Freeman N.J., Carrington S., Ronan G., Barrett P., Quantifying Structural Changes and Stoichiometry of Protein Interactions Using Size and Density Profiling. Letters in Peptide Science (2003) 10 487-494.
8. Using the van't Hoff and Eyring equations.
9. Boudjemline A., Clarke D.T., Freeman N.J., Nicholson J.M., Jones G.R., Early stages of protein crystallization as revealed by emerging optical waveguide technology J. Appl. Cryst. (2008). 41, 523-530. doi:10.1107/S0021889808005098.
10. Mashaghi A., Swann M., Popplewell J., Textor M., Reimhult E., Optical anisotropy of supported lipid structures probed by waveguide spectroscopy and its application to study of supported lipid bilayer formation kinetics, Anal. Chem., 80 (10), 3666-3676, (2008). PMID: 18517221 Web Release Date: 19, Apr.(2008); (Article) DOI: 10.1021/ac800027s.
11. Sanghera N., Swann M.J., Ronan G., Pinheiro T.J., Insight into early events in the aggregation of the prion protein on lipid membranes, Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1788, Issue 10, October (2009), Pages 2245-2251.

Copyright, Dr. Gerry Ronan (Farfield Group)

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