3. Dynamic Light Scattering in situ in common Crystallization Environments
3.7. In situ DLS: Overview, Conclusions and Outlook
53 be the cause of the distortion. Since not all drops were perfectly formed and the relative position of laser towards the droplets depends on the position of the well on the plate mechanical drift may not affect all wells. Thus the distortion can be explained.
If requirement b) for automation could be achieved in the course of experiments regarding in situ DLS in small capillaries (see chapter 3.2). The capillaries within the GCB-D only have an inner diameter of 100 µm. It is in this environment critical to be continuously on the same position. Even a deviation of just 20 µm will lead to a complete decline of the DLS signal. DLS was recorded at seven positions along a 100 µm inner diameter capillary during a counter diffusion experiment. The capillary was filled with glucose isomerase (50 mg/mL, in 0.1 M Hepes, pH 7.0), 3 M solution of ammonium sulfate was used as counter diffusion precipitant. In two weeks a total of 1022 measurements was recorded (146 per position), thus plate and optical head were moved 1022 times. It can be clearly seen from the results in chapter 3.2.3 that at positions at which no crystals grew within two weeks a valid ACF could be obtained throughout the experiment. This proves that automated in situ DLS measurements even in such critical environments as a 100 µm diameter glass capillary are possible.
3.6.4. Conclusions
The first automated DLS measurements within crystallization plates and thin capillaries could be carried out and it could be shown that even in the case of the GCB-D where highest accuracy is required automated monitoring of crystallization processes could be carried out. These results show that the integration of in situ DLS as a standard method within high throughput screening is possible and can be used to score and analyze crystallization droplets. However it is still required to adjust the position of laser and detector manually at all positions prior to the measurement. This needs to be automated as well in the future.
54 For the first time ever in situ DLS monitoring within Terazaki plates under oil, in 100 µm inner diameter capillaries within the Granada crystallization box domino (GCB-D) and in the channels of the CrystalFormer HT was established. Moreover the initial experiments in 96well-plates carried out by ARNE MEYER [92] could be improved.
Choosing seals (AMPLIseal, Bio Greiner One) and the plates best suited for in situ DLS (MRC Crystallization Plate™, Swissci) a better signal to noise ratio could be achieved and even more important: DLS is now possible in all 192 wells of the plate making it possible in the future to integrate in situ DLS as a standard step in high throughput screening, just as today imaging is carried out. The information gained by DLS surely will help to refine the crystallization experiments and facilitate optimization of crystal growth in order to obtain X-ray suitable crystals. However, application of in situ DLS towards different target proteins showed the complexity of this approach: in most cases the derived radius distributions over time showed a very complex pattern that was not comparable with the results obtained for model proteins (own results and results obtained by ARNE MEYER [92] and KARSTEN DIERKS et al. [88]). This opens the question if the use of model proteins leads to any scientific progress if it comes to the development of novel methods.
What target proteins often show (in the course of this work it was observed for SlfB (see chapter 5) and ΔN) is precipitation before crystallization. In these cases crystals grew out of the precipitate. If the conditions of the solution are altered to prevent precipitation no crystals appear at all. The application of in situ DLS could not reveal why this is the case.
Especially for in situ DLS within vapor diffusion crystallization experiments the shrinking drop size is a problem. An initially perfectly adjusted position of measurement can due to changes in the drop size eventually become a position at which DLS measurements are not or not good possible due to reflections and flares of the laser. The interpretation of data after such an experiment is then very difficult since CONTIN produces always a radius distribution and if the ACF is not interpreted manually such false positive results could easily be taken for real.
Promising are in situ DLS measurements in capillaries. Here the environment is stable; a once adjusted position will stay adjusted during the whole experiment.
Hence interpretable results can be obtained. Based on reflections of WILLIAM WILSON
[63] that if nucleation can be detected by DLS already to many nuclei exist in solution (which is not desired [40]) and of PETER G. VEKILOV that nucleation rather occurs
55 from a dense protein phase called the mesoscopic phase than directly from the dilute solution of protein molecules [47] monitoring of crystallization processes was focused to the analysis of the development of the RH of monomeric (or low oligomeric) particles. The same approach was proposed by AARON STREETS [93] and within the CrystalFormer HT comparable results, as first described by him in 2010, could be obtained for the time dependent development of RH in the case of a crystallizing and non-crystallizing systems.
In the course of this work the crystallization of various target proteins could be improved by in situ DLS:
• Jack Bean Urease [123] was obtained from Sigma as lyophilized powder for structure-function relation studies with inhibitors by AFSHAN BEGUM (University of Hamburg). All crystals only showed diffraction to approx. 3.5 Angstroms. In situ DLS revealed that the protein was oligomerized and that this oligomerization could not be removed. AFSHAN BEGUM then purified Jack Bean Urease from Jack Bean meal and could then grow crystals from the naturally occurring hexamers. These crystals proved to be X-ray suitable and showed diffraction up to 1.8 Angstroms. Inhibitor studies by AFSHAN BEGUM are in progress.
• WbGST, a glutathione-s-transferase from Wucheria bancrofti [124] was expressed and purified by PRINCE PRABHU (Centre for Biotechnology, Anna University, Chennai). No crystals could be obtained. An analysis of the most promising conditions and of variations of these conditions by in situ DLS showed rapid aggregation taking place inside the crystallization droplet.
Analysis of the protein without precipitant showed that its stability was highly temperature dependent. All crystallization processes were then carefully carried out at 4°C. X-ray suitable crystals could be obtained and the structure was solved by PRINCE PRABHU (to be published).
• ΔN, a recombinant protein construct of full length yeast ERV1 which is a sulfuryl hydroxylase [125], was investigated in terms of a cooperation with KYRIAKOS PETRATOS from IMBB Forth (Crete). Petratos and co-workers could only obtain tiny crystals. In situ DLS revealed that the used PEG concentrations were too high. Applying in situ DLS crystallization conditions were altered rationally. Reducing the concentration of PEG 4000 from 20% to 5% and addition of 5-8 % of PEG 400 resulted for the first time in the growth of
56 larger (300 – 400 µm) crystals that showed diffraction pattern. However the conditions need to be further optimized.
• The Spiegelmer NOXE36 was developed by NOXXON (Berlin, Germany) as an inhibitor of the monocyte chemoattractant protein (MCP-1) [126]. In the course of her scientific work BARBORA SCHMIDT (University of Hamburg)aimed at crystallizing the complex of NOXE36 and MCP-1. In situ DLS studies revealed that the initial crystallization conditions resulted in aggregation short after preparation of the crystallization experiments. Improvement of conditions led to the growth of X-ray suitable crystals that showed diffraction up to 2.2 Angstroms. Solution of the three dimensional structure is on-going.
Figure 30: A) Erv1 from Arabidopsis Thaliana (PDB-accession code: 2HJ3) as an example for sulfuryl hydroxylases. A recombinant fragment of Erv1 – ΔN – was crystallized with the help of in situ DLS. B) PfGST (PDB-accession code: 1PA3, [102]) as an example for Glutathione S-transferases such as WbGST from Wucheria bancrofti. The structure of WbGST was solved by the method of molecular replacement with PfGST as search model (33% sequence identity). C) Cartoon plot of the asymmetric unit of Jack bean urease (PDB-accession code: 3LA4, [123] ), the assumed biological molecule is composed of six monomers.
Three proteins were investigated as part of the EU FP6-funded OptiCryst [18]
consortium. The results of these investigations are presented as studies on the application of in situ DLS on target proteins in chapters 5 and 6.
• CD81 and CD82 two human membrane proteins of the Tetraspanin-family [127], were expressed and purified by ROSLYN BILL and NICKLAS BONANDER
(Aston University, UK). Applying in situ DLS crystals of both CD81 and CD82 could be obtained. Moreover interaction and oligomerization between and of CD81 and the tight junction protein Claudin-1 [128] was investigated by in droplet DLS. These studies are thoroughly discussed in chapter 6.
• SlfB from Lysinibacillus spaericus strain JGA12 [129], was purified directly from JGA12 by JOHANNES RAFF (Helmholtz-Zentrum Dresden-Rossendorf, HZDR) and co-workers. In situ DLS was applied to optimize buffer conditions
57 (chapter 5.3.1), investigate initial crystallization experiments (chapter 5.3.4) and to evaluate the influence of bivalent cations on the stability of SlfB (chapter 5.3.7).
In conclusion it can be stated that in situ DLS has a high potential to facilitate the rationalization of protein crystallization and to gain further insight into the submicroscopical processes within crystallizing solutions of biological macromolecules. In future light scattering methods in situ and prior to crystallization will help delivering the desired nano-crystals for structure elucidation of target proteins with X-ray lasers such as the XFEL currently being built at the DESY site.
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