Alteraciones de la respuesta emocional

4.1.1 S. cerevisiae Wpl1259-647 crystallisation

The WAPL domain stretches approximately from residue 259-647 in the S. cerevisiae Wpl1 (hereafter called ScWpl1259-647) and this construct was used for the initial crystallization trials. The protein was purified as described earlier in Experimental methods section. The crystal screens were set-up as 400 nL sitting drops containing equal volumes of the well-solution and the protein at 10 mg/mL in 96-well plates, which were then incubated at 4 ˚C. An initial hit was obtained in the condition 0.1 M BisTris Propane pH 6.5, 0.2 M Na-citrate and 20% PEG 3350. The crystals observed in the screen were very small, polygonal shaped and needed further improvement in both size and quality. Optimization was performed using 24-well hanging drop plates in which 4 µL drops were set by adding equal proportions of well-solution and protein at 10 mg/mL concentration. After optimization, the condition in which bigger crystals were obtained was 0.1 M BisTris Propane pH 6.5, 0.2 M Na-citrate and 15% PEG 3350. These crystals were much bigger (30x30x10 µm) compared to the initial ones in the screen but when tested on an in-house X-ray source, diffracted only to about 8.5 Å (Figure 4-1). To further improve these crystals, a multivariate screen was performed, in which the protein (8 to 15 mg/mL) along with salt (0.1 to 0.5 M) and precipitant (10 to

25%) concentrations were varied together within a narrow range around the original concentrations. After this optimization, bigger crystals could be obtained when the salt and protein concentrations were slightly increased (0.3 M Na-citrate and 12 mg/mL protein) while keeping precipitant concentration constant (15% PEG 3350). The biggest crystals that could be obtained were of dimensions 50x50x15 µm but when tested, the

3Å 4Å 6Å 12Å a _b 50 μm

Figure 4-1: Crystals and diffraction of ScWpl1259-647

diffraction only marginally improved to about 7 Å. Among other strategies that were tried were micro-seeding, dehydration, annealing and optimization of the cryoprotectant, none of which could adequately improve the diffraction of these crystals.

In order to get crystals belonging to a different crystal system, the following approaches were taken: (a) Change in the construct – several constructs starting from different nearby residues to the starting residue (E259) of the original construct were tried but these did not result in better crystals. (b) Different orthologues of Wpl1 – among the ones tried were human, Drosophila and Ashbya WAPL domains. The human and

Drosophila WAPL domains failed to crystallize while the one from Ashbya gossypii did

4.1.2 A. gossypii Wpl1184-561 crystallisation

Ashbya gossypii is a filamentous fungus whose genome is similar to that of budding

yeast with which it shares a common evolutionary origin. Around 95% of A. gossypii genes have orthologues in budding yeast and thus this organism provides a suitable alternative to S. cerevisiae for studying eukaryotic proteins. A sequence alignment of A. gossypii and S. cerevisiae Wpl1 proteins is shown in Figure 4-2.

In A. gossypii, the Wpl1 orthologue (AgWpl1) is slightly shorter in length, with 561 amino acid residues, compared to 647 in its budding yeast counterpart, with the WAPL domain extending from residues 184-561 (henceforth AgWpl1184-561). The purified AgWpl1184-561 was used to set up crystallization screens in 96-well sitting drop plates and 400 nL drops were set by adding 200 nL well-solution to an equal volume of the protein at 10 mg/ml concentration. Small crystals appeared within 2-4 days of setting up the trays in most of the screens across many different conditions. The crystals formed clusters of very small and thin plates stacked against each other through their flat surfaces (Figure 4-3). The best screen condition that produced crystals consistently when tested in 24-well plates using hanging drop method, was 0.02 M Na/K phosphate, 0.1 M Bis-tris propane pH 7.5, 20% PEG3350. The condition was further refined to 0.1 M Na/K phosphate, 0.1 M Bis-tris propane pH 7.5, 15% PEG 3350 and the protein concentration in the drops was reduced to 4 mg/mL to obtain bigger clusters. In order to obtain single crystals, several strategies were tried which included buffer and pH variation, screening for additives and microseeding. None of these techniques yielded desired results, so the individual plates had to be separated out carefully from the clusters to collect diffraction data. The Seleno-methionine (SeMet) - derivatised crystals were also grown in the same condition and formed clusters from which the individual plates had to be separated for mounting. The crystals were flash-frozen after soaking briefly in a solution containing 20% glycerol added to the mother liquor before mounting. The individual crystals diffracted to 2.8 to 3 Å in-house and about 2 Å in the synchrotron X-ray source (Figure 4-3). Both native and anomalous data were collected in the Diamond Light Source IO2 beamline. The structure solution process is discussed in the next section.

*:.**::* * .* .. * **.** * * ..* . *:::. . * :*. *: .::*:.: ::. .::**: . :.:::*** gi|374105930|gb|AEY94841.1| MPMKVYGRHGRYNRLVTTFGKTHGEE-DSWFSSDDENHGRPVSDDTTKLSLSQDRCSELPEETIGAN---RKRPAERTQTDP---VWEFLERPASGQKRRRR-- 95 gi|6320217|ref|NP_010297.1| --MRAYGKRGPVLRTPFRSNKGLPSSSDVEFSDDDVNSVIPDVSSTISSSIADHPIEGLLDEPRKAQDSSSSFDGANEKPSSQLDSKRNDQNVKIITSSDTSMAFMKDEKLSAFNFLDGSKASKRKRRRTY 129 ::. ..: ::. * : * .: :: * ..:: ::::*: :::* * . .. : *** **:* : ::: ::* : : .:..* : . : :.*:*** gi|374105930|gb|AEY94841.1| ---ATCDSTEYRESASQEFLNAVNVVQGIVSSLKP---AKEVVEHWAELEDVPEYR--GNNGQAVYGKTRTMLAKAEED---SDTEAAAHESDERAAQGDEALSRHFNEL 194 gi|6320217|ref|NP_010297.1| QKHDANITSSIEPDVQDEDSITMHNEFESIRKIYNDINEFILKLPRADDDILNKMLENEMKMDDSIENNSIRTSKDKKYGKFRTILINKNKENEIMGEEVDQKANTLSLNNADNSNAEKEGLTSTNHYNEL 260 :.**:*:**.:*::*:** **: .. :. :::*.*.* ::*:*:: ** :* ::*: : .* .. *** :: .*:. : * *::** : *:.*: :. . .:* :** gi|374105930|gb|AEY94841.1| RTMGETLKYSEDLDFILS---DNSMTTPEHRRNNMLRLCLDMMNNEDLCQYIVKYRHREVWEWCFQGTDPKQKVTSLLQCFIADKIPLLRHDKRWAMLSLENFILPLATDEVFPKKIAG---SRLVKLN 317 gi|6320217|ref|NP_010297.1| KNMGDTIKYQDDIEFLLSNSKSNDNTTVPINEYFKKLLNLSLMIINDEEFFQYAKRYFKKEIIKLSFAQFRSDFPELILLQGYLLHKVSESQSD---FPPSFDNFSIELSKDDGKIRTKKNKHIKKLSHLN 388 ::*:*** :*.. *:* :* : : : :. ** . :. .* .***: *.*: * .: *::* . .:::.: ..***: * :*: * . . : * : gi|374105930|gb|AEY94841.1| YQDLLRKLKFTNTCEYALYIWATYLLYTDAVYGAVPALARLISRGQLKDWDTACSLLENNIVAAPSGSDIEEYAQAFQTLAGLSREKLTNEGVLKCLIKLTNHTTVLELSADLLPSLVRSLAMSVQLHQNN 448 gi|6320217|ref|NP_010297.1| FEDFLRKTQFKTGLYYSLSLWEMHGNLSLDIIKRISILASNKD-LFSRHVKTFIPLLEKLITASEFCHMYIEQPEMFDSLISNLNNQFKDMLDDDSLIKILILLTNMEVHNYTLWKEADMIFQSSMNTILE 518 : .::: *.: ::*:*** *** *. .: . : :: ::* .:: . . ** :* ** * ::: . :* *.* ::* ***** ::.*.:*..* .:* .*: * gi|374105930|gb|AEY94841.1| IVSSISEIKTNLLILQLGLLLNIVSEATT--AASTEELTNFGAVFRSVFVKKP-TEMSFVLQLFLLVYAYSAG--AAGVQLPPAEADFLKSELEAFATDVSSYNHNIHTRITRVLETL--- 561 gi|6320217|ref|NP_010297.1| SIHPLTDAKVDNILLHLGLCLNICSRENSRLKLDGKLWYDMKTIFVKMIRDGSDTENRLVQGLFYLNFSFLIKQRKENSNLDPGELNLLLVELEAFKSETSQFNEGISNKIEIALNYLKSIYTSERITI 647 WPL!_AGOSS WPL!_SCERE WPL!_AGOSS WPL!_SCERE WPL!_AGOSS WPL!_SCERE WPL!_AGOSS WPL!_SCERE WPL!_AGOSS WPL!_SCERE

Figure 4-2: Alignment of Wpl1 sequences of S. cerevisiae and A. gossypii

Figure 4-3: Crystals and diffraction of AgRad61184-561

Crystals obtained in (a) initial screens and (b) after optimization. Diffraction images obtained with (c) in- house and (d) synchrotron X-ray sources. The edge of the synchrotron diffraction image shown in (d) is ~1.9 Å

4.2 AgWpl1

structure solution