Molecular Interactions

Analytical Ultracentrifugation (AUC) is an important group of powerful methods (sedimentation equilibrium and velocity) which allow determining the size, the approximate overall shape and the degree of homogeneity of proteins and other biological macromolecules in solution. These methods are specially adapted for the detection and quantitative analysis (proportion of species, stoichiometry, affinity, reversibility) of interactions leading to the formation of macromolecular complexes, including protein-protein, DNA-protein, and receptor-ligand interactions [Howlett et al. (2006) Curr. Opin. Chem. Biol. 10:430-436; Lebowitz et al. (2002) Protein Sci. 11:2067-2079; Minton (2000) Exp. Mol. Med. 32:1-5; Rivas et al. (1999) Methods 19:194-212].

Since its foundation in 1994, this CIB Facility has given scientific and technical support in the biophysical characterization of proteins and their interactions to a large number of research groups in the CIB, CSIC other centres, universities and other centres of domestic and foreign research. Moreover, the Facility counts with the advice and collaboration of international experts in the field and has participated in more than 300 publications since 1994.

Dynamic Light Scattering (DLS) provides complementary information to AUC, with two main advantages, relative to other commonly used analytical methods: low sample volume requirements (15-45 µl) and quick delivery of results (<10 min).
DLS allows determining the degree of homogeneity of macromolecular complexes in solution, to measure the translational diffusion coefficient of macromolecules and to calculate their hydrodynamic radius. This instrument (DynaPro MS/X model, Wyatt Inc.) is very well designed to study proteins and macromolecular assemblies (including polymers and liposomes), has a very easy operation (cuvette) and a Peltier temperature regulation system (4-60°C) and can be used to analyze different types of macromolecules such as proteins, polysaccharides, nanoparticles and lipid micelles.

Analytical Size Exclusion Chromatography coupled to Multi-Angle Light Scattering (SEC-MALS) separates the different macromolecular species present in the sample according to their size, and measures simultaneously their concentration and the intensity of the light scattered by these species, allowing the obtaining of their molecular weight.

For the rapid detection and real-time determination of the kinetic and equilibrium properties of homo- and hetero-associations of biological macromolecules and complexes in solution is also possible to use this instrument with a Composition Gradient (CG-MALS). This technique is based upon the methods of static light scattering recently developed by the group of Dr. Allen Minton, NIH [Attri & Minton (2005) Anal. Biochem. 337:103-110]. Main module is a multi-angle laser light scattering detector equipped with a flow cell (DAWN EOS model, Wyatt Inc.) that is connected to a refractive index flow detector and to a triple-syringe programmable pump. The information is acquired in minutes (important when the stability of the sample is a factor to consider). Moreover, the system allows determine the association/dissociation kinetics of a given macromolecular reaction (providing that the relaxation time of the reaction is in the minute range).

The Facility has carried out several studies on the oligomerization and polymerization of proteins by the combination of ultracentrifugation and light scattering techniques (Monterroso et al. (2013) Methods 59: 349–362; del Castillo et al. (2011) Biochemistry, 50: 1991–2003).

Biolayer Interferometry (BLI) is a simple, optical dip-and-read assay system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and lipids in real time and small sample volumes (4.5 µL). BLI provides direct binding affinities and rates of association and dissociation between a molecule, immobilized on a fiber-optic biosensor, and the analyte in solution. Since it requires nanomole amounts of sample, it is an excellent choice for the study of proteins that are challenging to isolate. BLI also has the advantage of quantifying protein concentration from heterogeneous crude lysates, since refractive index changes in the medium do not affect shifts in the interference pattern

Fluorescence Correlation Spectroscopy (FCS) allows evaluating the temporal fluctuations of the fluorescence intensity to obtain insight into the mobility of the molecules, which is related with their size and shape. In this way, protein polymerization and aggregation can be studied. FCS is also a powerful tool for the detection and measurement of molecular interactions rendering complexes substantially bigger than the free species. FCS constitutes an ideal complement for other methodologies available in the facility, such as analytical ultracentrifugation and light scattering (Monterroso et al. (2013) Methods 59: 349–362).

Fluorescence Intensity and Anisotropy/Polarization measurements allow the detection and quantification of molecular interactions such as those involving proteins, nucleic acids, peptides or ligands with high sensitivity and low sample consumption.  Anisotropy is particularly suitable for this purpose, as it depends on molecular volume and hence, it is sensitive to changes in size that occur upon complexation. The high sensitivity of these measurements (picomolar) enables evaluation of high affinity interactions under true equilibrium conditions. By using a plate reader, measurements are fast and can be performed simultaneously on hundreds of samples of low volume (a few microliters), opening the possibility of high throughput screening applications of interest in biotechnology and biomedicine.

Publications

Research Groups using the Facility

2023 Satisfaction Survey.