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Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering.

Oberthuer D, Melero-García E, Dierks K, Meyer A, Betzel C, Garcia-Caballero A, Gavira JA - PLoS ONE (2012)

Bottom Line: Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes.Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation.Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of infection and inflammation, University of Hamburg, Hamburg, Germany.

ABSTRACT
In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

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Graphical representation of a typical counter-diffusion experiment set-up in a GCB-Domino (Triana S&T).The dimension of the GCB-Domino is approximately 70 mm high × 17.2 mm wide × 7.0 mm thick. The open end of the capillary allows the precipitant (in blue) to diffuse against the much slower diffusive protein solution, thereby inducing the precipitation of the latter. The capillaries are kept in place by an agarose plug sitting at the top of the precipitant solution. Each mark (*) shows a DLS measurement position, at 0.7, 2.2, 5.1, 9.9, 11.8, 18.7 and 24.5 mm from the open end.
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pone-0033545-g004: Graphical representation of a typical counter-diffusion experiment set-up in a GCB-Domino (Triana S&T).The dimension of the GCB-Domino is approximately 70 mm high × 17.2 mm wide × 7.0 mm thick. The open end of the capillary allows the precipitant (in blue) to diffuse against the much slower diffusive protein solution, thereby inducing the precipitation of the latter. The capillaries are kept in place by an agarose plug sitting at the top of the precipitant solution. Each mark (*) shows a DLS measurement position, at 0.7, 2.2, 5.1, 9.9, 11.8, 18.7 and 24.5 mm from the open end.

Mentions: The crystallization condition employed by Carrel and co-workers [44] to crystallize glucose isomerase (solved its structure at 1.9Å) was slightly modified to fulfil the requirements of counter-diffusion experiments [45] by increasing the original concentration of ammonium sulphate from 0.7 M to 3.0 M. Hence, Granada Crystallization Boxes-Domino (GCB-D) reactors [35] containing ammonium sulphate (3.0 M) and Hepes (0.1 M) at pH 7.0 were purchased from Triana S&T and used to set up the crystallization experiments. The GCB-Ds are usually topped with a layer of agarose gel for mechanical stability when the capillaries are punctured (see Figure 4). Then, capillaries of 0.1, 0.2 and 0.3 mm inner diameter were filled with protein stock solution. An extra fourth capillary of 0.3 mm was filled with a mixture of protein solution, buffer and 0.075% w/v low melting point agarose (AG-LGT, Triana S&T). All DLS experiments were carried out at RT.


Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering.

Oberthuer D, Melero-García E, Dierks K, Meyer A, Betzel C, Garcia-Caballero A, Gavira JA - PLoS ONE (2012)

Graphical representation of a typical counter-diffusion experiment set-up in a GCB-Domino (Triana S&T).The dimension of the GCB-Domino is approximately 70 mm high × 17.2 mm wide × 7.0 mm thick. The open end of the capillary allows the precipitant (in blue) to diffuse against the much slower diffusive protein solution, thereby inducing the precipitation of the latter. The capillaries are kept in place by an agarose plug sitting at the top of the precipitant solution. Each mark (*) shows a DLS measurement position, at 0.7, 2.2, 5.1, 9.9, 11.8, 18.7 and 24.5 mm from the open end.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366972&req=5

pone-0033545-g004: Graphical representation of a typical counter-diffusion experiment set-up in a GCB-Domino (Triana S&T).The dimension of the GCB-Domino is approximately 70 mm high × 17.2 mm wide × 7.0 mm thick. The open end of the capillary allows the precipitant (in blue) to diffuse against the much slower diffusive protein solution, thereby inducing the precipitation of the latter. The capillaries are kept in place by an agarose plug sitting at the top of the precipitant solution. Each mark (*) shows a DLS measurement position, at 0.7, 2.2, 5.1, 9.9, 11.8, 18.7 and 24.5 mm from the open end.
Mentions: The crystallization condition employed by Carrel and co-workers [44] to crystallize glucose isomerase (solved its structure at 1.9Å) was slightly modified to fulfil the requirements of counter-diffusion experiments [45] by increasing the original concentration of ammonium sulphate from 0.7 M to 3.0 M. Hence, Granada Crystallization Boxes-Domino (GCB-D) reactors [35] containing ammonium sulphate (3.0 M) and Hepes (0.1 M) at pH 7.0 were purchased from Triana S&T and used to set up the crystallization experiments. The GCB-Ds are usually topped with a layer of agarose gel for mechanical stability when the capillaries are punctured (see Figure 4). Then, capillaries of 0.1, 0.2 and 0.3 mm inner diameter were filled with protein stock solution. An extra fourth capillary of 0.3 mm was filled with a mixture of protein solution, buffer and 0.075% w/v low melting point agarose (AG-LGT, Triana S&T). All DLS experiments were carried out at RT.

Bottom Line: Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes.Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation.Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of infection and inflammation, University of Hamburg, Hamburg, Germany.

ABSTRACT
In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

Show MeSH
Related in: MedlinePlus