Biomedical subjects

Emmanuel Saridakis

Publications and source records attributed to Emmanuel Saridakis.

3 recordsLinked to original sources

PubMed2003

Systematic improvement of protein crystals by determining the supersolubility curves of phase diagrams.

A systematic approach for improving protein crystals by growing them in the metastable zone using the vapor diffusion technique is described. This is a simple technique for optimization of crystallization conditions. Screening around known conditions is performed to establish a working phase diagram for the crystallization of the protein. Dilutions of the crystallization drops across the supersolubility curve into the metastable zone are then carried out as follows: the coverslips holding the hanging drops are transferred, after being incubated for some time at conditions normally giving many small crystals, over reservoirs at concentrations which normally yield clear drops. Fewer, much larger crystals are obtained when the incubation times are optimized, compared with conventional crystallization at similar conditions. This systematic approach has led to the structure determination of the light-harvesting protein C-phycocyanin to the highest-ever resolution of 1.45 A.

Crystallization↗

PubMed2002

Separating nucleation and growth in protein crystallization using dynamic light scattering.

A means of controlling crystallization is to separate the phases of nucleation and growth. Methods to achieve this, other than seeding, involve lowering the supersaturation by changing the temperature or diluting drops after incubating them for a given time at nucleation conditions. However, by the time nuclei or crystals are visible under the microscope too many nuclei will have formed. Dynamic Light Scattering was applied practically, to determine the most likely time for nucleation-growth decoupling to be performed successfully. The time at which DLS showed a significant change in the size-distribution of species in solution, corresponded to that optimal time.

Animals↗

PubMed2002

Protein crystallization for genomics: towards high-throughput optimization techniques.

Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X-ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X-ray analysis and bioinformatics stages once diffraction-quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large-scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high-throughput techniques.

Automation↗