High-throughput SRCD using multi-well plates and its applications

Abstract

The sample compartment for high-throughput synchrotron radiation circular dichroism (HT-SRCD) has been developed to satisfy an increased demand of protein characterisation in terms of folding and binding interaction properties not only in the traditional field of structural biology but also in the growing research area of material science with the potential to save time by 80%. As the understanding of protein behaviour in different solvent environments has increased dramatically the development of novel functions such as recombinant proteins modified to have different functions from harvesting solar energy to metabolonics for cleaning heavy and metal and organic molecule pollutions, there is a need to characterise speedily these system.

Circular dichroism studies of low molecular weight hydrogelators: The use of SRCD and addressing practical issues

“…To assess whether LD, LB, and CB contributions were negligible or dominant, synchrotron radiation circular dichroism (SRCD) measurements can be conducted for several small spots, 0.5 mm in diameter, along the hydrogel specimens. The use of the vertical sample chamber of the B23 beamline made it possible to measure a hydrogel laid horizontally, without the complication of thickness due to a change in gravity (Figure ) …”

“…The use of the vertical sample chamber of the B23 beamline 33 made it possible to measure a hydrogel laid horizontally, without the complication of thickness due to a change in gravity ( Figure 3). 34 Extensive optimization of both the sample preparation and handling enabled the development of a series of experiments to (i) identify the presence of optical artifacts (LD and LB), (ii) observe the homogeneity of the samples, and (iii) monitor both the thermal disruption and reformation of the hydrogels (sol-gel reversibility). Due to the time constraints of beamtime for this study, spectra were only acquired at rotations of 0°and 90°; therefore, only LD and LB were evaluated for these samples.…”

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

“…Many research groups have used SRCD spectroscopy to examine and estimate the secondary structure content of proteins (Lees et al 2006;Matsuo and Gekko 2013;Garcia et al 2013;Hussain et al 2016;Lopes et al 2016;Miler et al 2016). The technique has been successfully employed to characterize the insertion and orientation of helical peptides in lipid bilayers (Bürck et al 2016) or compare the behavior of globular and natively unfolded proteins (Ruskamo et al 2012;Yoneda et al 2017), investigate protein/peptide structure in model membranes (Miles et al 2008;Dreschler et al 2009), assess the dynamic and flexible conformation of unordered proteins (Lopes et al 2014a, b;Bremer et al 2017), evaluate protein:protein complex formation (Cowieson et al 2008;Lopes et al 2013), detect conformational changes induced by the binding to ligands (Lima et al 2013), perform fold recognition and obtain an accurate distinction between parallel and antiparallel β-sheets (Micsonai et al 2015) and identify the conformational changes associated with a mutant protein (Wallace et al 2004).…”

Characterisation of sensor kinase by CD spectroscopy: golden rules and tips

“…Most importantly, such an increased brightness is delivered as a highly collimated microbeam with a diameter that can vary from 1 to 0.05 mm compared with 8–3 mm for conventional benchtop CD instruments. This unique feature enables the study of comparatively smaller sample volumes for a variety of microcuvette cells, flat capillaries and the use of multiwell plates (Figure 3) [52].

Figure 2.Schematic of the vertical chamber available at B23.( A ) Diagram of the arrangements of optical elements in the vertical chamber leading to the sample chamber ( B ) housing an X–Y motorised stage which is able to hold a 384- or 96-well plate as shown in ( B ).

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Figure 2.Schematic of the vertical chamber available at B23.( A ) Diagram of the arrangements of optical elements in the vertical chamber leading to the sample chamber ( B ) housing an X–Y motorised stage which is able to hold a 384- or 96-well plate as shown in ( B ). Image redrawn from Hussain et al [52]. 1, 0.02 and 0.001 cm fixed pathlength cells are available for 96-well multi-cells, and volume-dependent pathlengths are available to 384-well plate.

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