In situ Proteolysis of SlfB

Initially the Floppy Choppy-kit (Jena Bio Science) was applied for in situ proteolysis [163, 164] of full length SlfB from JGA12. This method was chosen after crystallization experiments only yielded small crystals even after extensive screening and optimization of buffer- and crystallization conditions. Moreover DLS and SAXS

83 measurements showed that SlfB has an elongated shape in solution (see chapter 5.2.5) which points to the possibility of highly flexible domains that – without further modification – would resist attempts to form large crystals. It could be shown [163, 164] that the method of in situ proteolysis provides a good way to get at least X-ray structures of single domains of large and/or flexible proteins.

In situ DLS monitoring of the proteolysis experiments was carried out directly after mixing SlfB and the respective protease solutions and afterwards in selected intervals during two weeks. DLS has the advantage over SDS-PAGE [157] that it directly yields information of the non-denatured status of the protein. Still SDS-PAGE was used to gain additional information on the state of proteolysis. For both DLS and SDS-PAGE undigested JGA12-N in its buffer and in the proteolysis buffer conditions were used as reference. In the course of the DLS monitoring conclusions were drawn and new in situ proteolysis experiments were started with larger volumes. In the initial experiments (see Figure 50) a fast decrease of RH (from the 6.8 nm of pure SlfB) could be observed at 100 µg/mL subtilisin (1:10 dilution), in all other solutions except subtilisin at 1:100 dilution and papain at 1:10 – where a slow decrease could be observed – the RH remained nearly constant. These observations hold true during the two weeks, the hydrodynamic radius of the sample digested by 100 µg/mL subtilisin decreased to approx. 3.8 nm while the intensity of scattered light vanished more and more. At this concentration it seems that subtilisin digests SlfB almost completely.

Figure 50: DLS monitoring of in situ proteolysis experiments for two weeks.

84 At a higher dilution of subtilisin (1:100 dilution, 10 µg/mL) the RH decreased slowly to about 5 nm after two weeks. If papain at 1:10 dilution (100 µg/mL) was used the RH

after two weeks was 5.5 nm. For all other in situ proteolysis experiments (papain at 1:100 dilution, trypsin and chymotrypsin at 1:10 and 1:100 dilution) no significant change of RH could be observed. Based on these initial results large scale proteolysis set-ups applying subtilisin at 1:100 and papain at 1:10 dilution were carried out.

Figure 51: SDS-PAGE to monitor in situ proteolysis of SlfB. Whilst digestion of SlfB with 1:10 trypsin shows only little change of RH in the DLS measurements it appears in the gel as two bands representing two fragments (Sub = subtilisin, Pap = papain).

Further analysis of these experiments by SDS-PAGE (Figure 51) showed that no fragments larger than 18 kDa were present in the digested sample in the case of digestions with papain and subtilisin, whilst in DLS particles with a RH between 4.5 and 5.5 nm could still be detected. The SLH domains of SlfB have an estimated MW

of 16 kDa, it is possible that proteolysis by papain of subtilisin leads to a digestions of all SlfB, but the SLH domains. Crystallization experiments were carried out with both (subtilisin 1:100 and papain 1:10) proteolysis solutions. The concentration of the samples was adjusted applying the pre crystallization test (PCT, Hampton) and both solutions containing SlfB digested by subtilisin at 1:100 and by papain at 1:10 dilution were screened against 480 conditions applying the Honeybee 961 robot. After three

85 weeks of incubation at 20°C crystals of SlfB digested by papain grew in condition C10 (10% w/v PEG 8000, 20% v/v ethylene glycol, 0.03 M of each NPS (sodium nitrate, disodium hydrogen phosphate, ammonium sulfate), 0.1 M bicine/Trizma base pH 8.5) of the MORPHEUS screen [165]. These crystals (Figure 52), however are too small for UV-analysis and X-ray diffraction and need further optimization.

Figure 52: Crystals of SlfB digested with papain (1:10 dilution). Crystals were grown in condition C10 (10% w/v PEG 8000, 20% v/v ethylene glycol, 0.03 M of each NPS (sodium nitrate, disodium hydrogen phosphate, ammonium sulfate), 0.1 M bicine/Trizma base pH 8.5) of the MORPHEUS screen.

Figure 53: Crystals of SlfB digested by subtilisin grown in solution A1 of the PCT (Hampton). The strong UV-fluorescence (B) shows that the crystals are protein crystals.

After two month crystals (Figure 53) of SlfB digested with subtilisin could be observed in the PCT drop A1, containing 2.0 M ammonium sulfate and 0.1 M TRIS

A) B)

86 hydrochloride (pH 8.5). UV-Imaging [119] of the crystallization droplet applying a CrystalLIGHT 100 (Nabitec, Germany) light source showed fluorescence of the crystals (Figure 53) indicating them being protein crystals. Crystals were analyzed at the consortiums beamline X13 (HASY- LAB / DESY) and proved to be X-ray suitable (see Figure 54). However the diffraction was too weak applying synchrotron radiation at DORIS III to estimate cell constants. Crystals are stored at 100 K for further investigation at the P11 beamline of PETRA III.

Since SDS-PAGE showed fragmentation of SlfB by trypsin (Figure 51) that could not be observed by DLS, a large scale approach of digestion with trypsin and chymotrypsin at 1:10 dilution was set-up. After one week of incubation the concentration of the samples was adjusted as described for the digestions with subtilisin and papain. The concentrated samples were screened against 480 conditions applying the Honeybee 961 robot. No crystals could be obtained.

Figure 54: Diffraction image of one of the crystals shown in Figure 53. However, the spots visible were too weak to index the crystals. But it proves that the crystals are indeed protein crystals.

To analyze the digestion of SlfB by trypsin and chymotrypsin further SAXS was applied. As described in chapter 5.3.5 SAXS data were processed by GNOM [152].

For digestion with trypsin at 1:10 dilution the radius of gyration (Rg) was determined to be approx. 5.9 nm (undigested: Rg = 6.13 nm), which is in concordance with the

Resolution limit: 3.3 A

87 DLS results showing only minor changes in RH. However the MW determined by SAXS is with about 62 kDa much smaller as for undigested SlfB (115 kDa) but similar to the MW obtained by SDS-PAGE. For digestion with chymotrypsin at 1:10 dilution Rg was determined to be approx. 5.9 nm thus being very similar to the digestion with trypsin. Also the MW determined by SAXS is approx. 64 kDa nearly identical with the results obtained for digestion with trypsin.

Table 3: In situ proteolysis of SlfB with trypsin and chymotrypsin, comparison of DLS and SAXS results.

RH (nm) (DLS) Rg (nm) (SAXS) MW (kDa) (SAXS)

SlfB 6.61±0.07 6.14±0.12 115

SlfB digested with

trypsin 6.26±0.13 5.93±0.18 62

SlfB digested with

chymotrypsin 6.14±0.09 5,92±0.27 64

DAMMIF [155] was used from the ATSAS online server [153] for ab initio modeling of the digested fragments of SlfB and comparison with the full length protein.

Alignments of the DAMMIF-models with PyMol show that digestion with trypsin results in a branched and elongated molecule bearing not many similarities with SlfB.

This is different for chymotrypsin here an alignment with full length SlfB shows that both molecules are nearly identical.

Figure 55: Alignment of DAMMIF models of (A) full length SlfB (blue) and trypsin digested SlfB (cyan) and (B) full length SlfB (blue) and chymotrypsin digested SlfB (grey).

The model obtained by DAMMIF from SAXS data for SlfB after digestion with chymotrypsin lacks only a terminal domain. It was proposed in chapter 5.3.5 that the here missing part of SlfB consists of the SLH domains. Thus one possibility is, that chymotrypsin digests these N-terminal domains of SlfB. However the MW of the remaining fragment is with – as determined by SAXS –approx. 62 kDa lower than the

A) B)

88 expected MW of SlfB without the SLH domains (approx. 100 kDa: SlfB: 115 kDa, SLH domains of SlfB approx. 16 kDa).