Molecular and systems biophysics

Accurate structural characterisation

Structure-function characterisation of biomolecules is vital as structure dictates function. Interaction between biomolecules or macromolecules are essential for all cellular processes and therefore characterising these interactions is an important task.

Circular dichroism

Circular dichroism (CD) is used to obtain structural details of any chiral molecules in solution, by measuring a difference in absorbance between left and right circularly polarised light. Common measurements performed at NPL include:

• Peptides
• Virus-Like-Particles (VLPs)
• Proteins including Monoclonal antibodies (mAbs)
• DNA/RNA

NPL led two inter-laboratory studies resulting in peer-reviewed publications reflecting the findings along with best-practise guidance for acquiring reliable and comparable measurements. NPL has also taken part in developing a new reference material for CD spectroscopy. Read more about this work:

• Val-CiD Appendix A: CD spectroscopy: an interlaboratory study
• Val-CiD Best Practice Guide: CD spectroscopy for the quality control of biopharmaceuticals
• A new reference material for UV-visible circular dichroism spectroscopy

Linear dichroism

Linear dichroism (LD) is used to obtain structural details of any chiral molecules in solution, by measuring a difference in absorbance between parallel and perpendicular linearly polarised light. LD is used to study long molecules such as DNA; fibrous proteins, such as collagen; and peptides/proteins interaction with liposomes.  This solution-based technique gives information that is unique and provides a rapid answer to mechanistic aspects of biological function which is not possible by NMR or X-ray diffraction due to the size and flexibility of these long molecules. Common measurements at NPL include:

• Peptide-membrane interactions
• Protein-membrane interaction
• DNA/RNA-small molecule interaction
• DNA/RNA-peptide/protein interaction
• Fibrous protein/Alzheimer and prion proteins

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is effective in identifying compounds by measuring mid-infrared light absorption yielding distinctive molecular fingerprints. FTIR of inorganic, organic or polymer chemical compounds can be acquired in powder or in solution phase. The method is used to study consumer products such as paints, polymers, coatings, fertilisers, food and pharmaceutical products. Common measurements performed at NPL include:

• Chemical compounds
• Chiral molecules

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) allows label-free characterisation of molecular interaction in solution, by direct measure of heat exchange during the interaction. Comprehensive information on molecular interaction such as affinity, stoichiometry and thermodynamics can be obtained by the raw heat pulses generated by ITC. Insight into binding at a molecular level can be gauged via the thermodynamic profiles, ΔH, ΔG, ΔS and steady state affinity via K_D. Common measurements include:

• Peptide-peptide interaction
• Peptide-protein interaction
• Protein-protein interaction
• Peptide/protein-membrane interaction
• DNA-peptide/protein interaction
• VLP-cargo encapsulation
• Enzymatic activity

NPL is leading a round robin study under VAMAS to validate the method using ITC characterising membrane-antimicrobial peptide interactions. 

These services are delivered using the following equipment:

• Jasco CD/LD- 1500
• UV-VIS (Perkin Elmer)
• Bruker FTIR (Tensor 27)
• ITC 200
• Applied Photophysics Chiroscan

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