Screening crystallisation conditions using fluorescence correlation spectroscopy

Abstract

We investigate the potential of fluorescence correlation spectroscopy (FCS) in screening for crystallisation conditions. Solutions that nucleate protein crystals must have different interactions than solutions that do not give rise to crystals. Due to these different interactions the average mean squared displacement of the individual proteins changes. By monitoring protein self-diffusion, we can distinguish crystallizing from non-crystallizing solutions. The method introduced can be applied at extremely low concentrations in femtoliter volumes as an early diagnostic for molecular association. Based on our preliminary findings FCS has the potential to become a routine screening method for crystallography.

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

“…Consequently, it was confirmed that our established protocol and FCS system is a useful tool for high‐throughput assessment of protein aggregation and screening for solution conditions for NMR samples. This method is also applicable for screening crystallization conditions in X‐ray analyses16 and assessing stability of target proteins in various solutions by altering the interval periods between sample preparation and FCS measurements. This system can also be expanded to a robotic system using an automated liquid handling machine.…”

Kinetics of Insulin Adsorption at the Oil–Water Interface and Diffusion Properties of Adsorbed Layers Monitored Using Fluorescence Correlation Spectroscopy

“…Crowding has previously been observed by FCS to influence the diffusion constant significantly (10). In bulk 3D diffusion, the diffusion constant decreases at protein volume fractions of only a few percent.…”

“…The dissociation constants and on/off rates of C-peptide to human cell membranes were measured by Rigler et al (8), and the dynamics of " 2 -adrenergic receptorYligand complexes was recently measured on living cells (9). Schmauder et al (10) demonstrated how FCS may be used to screen for crystallizing conditions in protein solutions. To our knowledge, diffusion of proteins at the oilYwater interface has not been previously studied via FCS, although it provides a model system for two-dimensional diffusion.…”

Heterogeneous nucleation of three-dimensional protein nanocrystals

“…To predict protein nucleation and crystallization, techniques such as dynamic light scattering (Berne & Pecora, 1976;George & Wilson, 1994) and fluorescence correlation spectroscopy (Schmauder et al, 2002) have been applied. Most studies based on these techniques have shown that the second virial coefficient B 22 of the dilute protein solution is closely related to protein crystallization (George & Wilson, 1994;Velev et al, 1998;Narayanan & Liu, 2003).…”