Peptides and proteins - characterisation

Characterization of peptides and proteins

Circular dichroism
CD measurements can be carried out both in the far-UV range (190-250 nm) and in the near-UV range (250-310 nm). The CD signal in the far-UV range is sensitive to the conformation of the peptide backbone angles and thus reports on the secondary structure. Proteins also contain groups that display near-UV CD activity, Trp, Tyr, Phe and, to a smaller extend,  S-S bonds. The near-UV signal is dependent on the chirality of the microenvironment of these chromophores and thus provides a spectral signature of the tertiary structure of the protein.

Dynamic Light Scattering
Dynamic Light Scattering (DLS) measures the hydrodynamic properties of solutes or small particles, which can be resolved to size distributions. Due to the size dependence of the scattering intensity, DLS is a very sensitive tool for the detection of minute amounts of multimers and aggregates.

Fluorescence spectroscopy
The fluorescence emission characteristics of side chain fluorophores of proteins are highly sensitive to the local environment of the fluorophores. Therefore, fluorescence spectroscopy can be used to monitor conformational changes of a protein or the binding of a ligand. A significant advantage of using fluorescence spectroscopy over near-UV CD is that generally much smaller amounts of protein are required. On the other hand, the complex nature of the fluorescence emission of protein fluorophores makes the interpretation of emission spectra challenging, especially if no complementary tool is applied.
Our fluorescence spectrometer is also equipped with polarizers, making steady state anisotropy measurements possible. The anisotropy of a fluorophore is dependent on the motional tumbling of a fluorophore and is therefore affected by binding events, including protein oligomerization.

Infrared spectroscopy (FTIR)
FTIR is a sensitive method to determine the protein secondary structure. It is especially sensitive to beta-sheet structures and in particular for intermolecular beta-sheet structures. However, the most important advantages in this context are the abilities to analyze both solid and liquid samples, the latter of highly concentrated aqueous solutions. FTIR can therefore be used to measure the secondary content of a protein under many formulation conditions. Measurements on dilute samples are also possible but need to be carried out in D2O.

Thioflavin T fluorescence
Thioflavin T is the most commonly used fluorophore probe for fibril formation  and can be used in situ to determine the fibrillation rate and, in some cases, to quantify the amounts of fibrils present in a sample.