SDS-PAGE and western smudge analyses have proved that all the FXII-RFPs have been successfully expressed. As it were the supernatants ( soluble fraction ) of the cell lysates that were purified, these analyses show that the FXII-RFPs are soluble. The homology theoretical accounts of FNII and EGF-1 sphere of FXII ( Figures 3.14 and 3.18 ) show that these spheres have got two and three disulphide bonds severally. Therefore, post-translational disulphide bonds formation is indispensable for the stableness of the FXII-RFPs and therefore their solubility. In state of affairss where disulphide bonds have failed to organize, the unfolded proteins formed are unstable and will either be found in inclusion organic structures of E.coli or undergo debasement ( Peisley and Gooley, 2007 ) . Hence, this survey has demonstrated that amalgamate Trx-tag has aided disulphide bonds formation in the FXII-RFPs taking to their solubility.
4.1.2 IMAC: An Analytical and Characterisation Tool
The construct of His-tag purification via immobilised metal affinity chromatography ( IMAC ) is that the histidine residues will adsorb strongly to the column based on interaction between Ni2+ and the electron-donating groups on histidines ( Figure 4.1 ) . The keeping of a protein in a metal ion column is influenced by the spacial distributions and handinesss of histidine residues on its surface ( Hemdan et al, 1989 ; Gaberc-Porekar and Menart, 2001 ; Ueda et Al, 2003 ) . Although histidine is a common amino acid residue in natural proteins, histidines on a protein surface will adhere to Ni2+ with somewhat lower affinity as compared to a flexible His-tag ( Gaberc-Porekar and Menart, 2001 ; Block et Al, 2009 ) .
Figure 4.1 A conventional representation of a His-tagged protein edge to metal-chelated affinity support. The construct of IMAC is based on the interaction between immobilised metal ions, in this instance nickel ions, which serve as the electron-pair acceptors and the amino acid residues on the protein surface, in this instance histidine residues, which serve as negatron givers. ( Adapted from Gaberc-Porekar and Menart, 2001 ) .
The FXII-RFPs in this survey were eluted at imidazole concentrations ( [ iminazole ] ) that range between 440nm to 510nm. These concentrations are instead high as compared to old study that 20-250mM iminazole is effectual for the elution of 6xHis-tagged protein ( Terpe, 2003 ) . In the homology theoretical accounts of FXII structural spheres ( Figure 4.2 ) , it was found that all the histidine residues in these spheres are exposed to the dissolver accessible surface, the functional surface with regard to the environing dissolver ( Ridgen, 2009 ; chapter 7 ) . Therefore, these surface exposed histidines can adhere to Ni2+ and accordingly lend to the high affinity of these FXII-RFPs for Ni2+ column.
( A )
( B )
( C )
Figure 4.2: Cartoon representations of the homology theoretical accounts of the assorted structural spheres of FXII demoing the dissolver accessible surface. The coloring material of the surface corresponds to the coloring material of the implicit in molecule. These theoretical accounts show that the histidine residues ( blue ) are exposed on the surface. ( A ) Homology theoretical account of FXII1-71.The N-terminal part of unknown homology ( aminic acids 1-22 ) is shown in purple, the FNII sphere ( aminic acids 23-71 ) is shown in bluish green ; ( B ) Homology theoretical account of FXII23-71, the FNII sphere ; ( C ) Homology theoretical account of FXII EGF-1 sphere ( aminic acids 75-112 ) .
Imidazole concentration % /mM ( at which proteins were eluted )
Number of histidine residues ( excludes His-Trx ticket )
11However, it is interesting that the [ imidazole ] required to elute the FXII-RFPs do non correlate with the figure of histidine residues. f-FXII23-112 and f-FXII23-154, which have more histidine residues as compared to f-FXII1-71 and f-FXII23-71, were eluted at lower [ imidazole ] ( Table 4.1 ) which indicates that f-FXII23-112 and f-FXII23-154 have lower adhering affinity to Ni2+ column as compared to f-FXII1-71 and f-FXII23-71. This may propose that the histidine residues in these larger proteins are less accessible for adhering to Ni2+ .
Table 4.1: Table shows the imidazole concentration at which the FXII-RFPs were eluted and entire figure of histidine residues in each FXII-RFPs.
It has been reported that IMAC can be exploited as an analytical tool to examine the topography of histidine residues of a protein molecule ( Hamden, 1989 ) . Therefore farther probes of the behavior of these FXII-RFPs in IMAC may uncover of import information in the spacial distribution of the histidine residues. As it has been reported that histidines play a major function in the surface adhering activity of FXII ( Samuel et al, 1993 ) , information about their distribution on the surface of FXII is important for the probe of FXII adhering to negatively-charged surfaces.
The chromatogram for f-FXII23-71 ( Figure 3.2B ) shows that its elution produced a individual crisp extremum. This indicates that the f-FXII23-71 proteins have similar affinity for Ni2+ which may propose that they have adopted a similar conformation. This is the same for f-FXII1-71, f-FXII23-112 and f-FXII23-154 ( Figure 3.2A, C, D ) . The deduction of this observation will be discussed further in subdivision 4.2.3.
4.2 EXPRESSED FXII-RFPs ARE MONOMERIC AND GLOBULAR
4.2.1 Analysis of Protein Elution via GFC
The rule of protein separation in GFC is based on the hydrodynamic volume of a protein, the volume which it takes up when it is in the solution. The pores in the gel filtration column service as molecular screens where the deceleration clip of a protein in a column will depend on its form every bit good as molecular weight ( Rosenberg, 2002 ) . Therefore, two proteins of similar MW which behave otherwise in solution may hold different hydrodynamic volumes and therefore different keeping clip in a column ( Rodr & A ; iacute ; guez-D & A ; iacute ; az et Al, 2005 ) .
In the chromatogram for f-FXII23-71 ( Figure 3.5B ) , a little prima extremum is seeable right before the crisp extremum produced by protein elution from the gel filtration column S75 GL. When the 2nd half of f-FXII23-71 was purified utilizing a different column, S75 PG, the presence of a little prima extremum can once more be noticed in the chromatogram ( Figure 3.7A ) . The difference between the above two mentioned columns is their bed volume. S75 GL has a bed volume of 24ml whereas S75 PG has a bed volume of 120ml ; column with a larger bed volume has a higher separation declaration. Therefore, it is improbable that this little prima extremum is caused by a contamination of different MW or f-FXII23-71 of a different conformation because S75 PG, which has a higher separation declaration, would be able to divide that contamination ensuing in a distinguishable extremum.
The chromatograms of f-FXII1-71, f-FXII23-71 and f-FXII23-112 ( Figure 3.6 and Figure 3.7 ) show that the elution of FXII-RFPs from gel filtration column produced individual crisp A280nm extremum. These show that the eluted FXII-RFPs were extremely homogenous.
4.2.2 GFC as an Analytic Tool
GFC has been widely used to analyze conformation alterations of protein folding and flowering ( Martensen, 1978 ; Bates et Al, 1997 ; Uversky et Al, 2001 ) because proteins of the same that are folded and unfolded will act otherwise in GFC due to their different hydrodynamic volumes. In this survey, the estimated MW of f-FXII1-71 and f-FXII23-71 were rather close to their theoretical MW ( Table 3.3 ) . As ball-shaped proteins of known MW were used for standardization of the S75 GL, this MW appraisal shows that f-FXII1-71 and f-FXII23-71 have got a ball-shaped form. In add-on, f-FXII1-71 was eluted with crisp individual extremum, proposing that they are of the same species with similar conformation. This is besides the same for f-FXII23-71.
These indicate that the amalgamate Trx was sufficient for disulphide bonds formation in the FXII-RPs resulting in all of them holding a similar ball-shaped conformation. The footing of this suggestion is that if the proteins were non folded, their estimated MW will be larger than what were obtained because unfolded proteins will follow a random spiral constellation and accordingly have a larger hydrodynamic volume as oppose to pack ball-shaped proteins.
Besides this, the estimated MW besides shows that f-FXII1-71 and f-FXII23-71 exist as monomers. The other column used in this survey, S75 PG, was non calibrated, therefore it is non possible to gauge the MW of f-FXII23-112. However, based on ( 1 ) the chromatogram ( Figure 3.7B ) which shows a crisp individual extremum ; ( 2 ) the difference in the Ve of f-FXII23-71 and f-FXII23-112 was non immense, it is improbable that f-FXII23-112 have formed sums.
4.2.3 Role of Trx in Disulphide Bonds Formation
Observations from both IMAC and GFC have provided strong grounds that the Trx-tag entirely is sufficient for post-translational disulphide bond formation in the FXII-RFPs.
In the cytol of E.coli, thioredoxin reductase ( the merchandise of the trxB cistron ) maintains thioredoxin 1 and 2 ( encoded by trxA and trxB severally ) in their decreased province. These thioredoxins will in bend cut down thiol groups of proteins in the cytol ( Stewart et al, 1998 ; Jurado et Al, 2006 ) . The cut downing environment is the chief ground why it is non possible for formation of disulphide bond in the E.coli cytol ( Peisley and Gooley, 2007 ) . It has been proposed that when thioredoxin reductase is absent, the thioredoxins will function as oxidant alternatively of their usual function as reducing agents. Therefore, the usage of Trx merger protein together with trxB deficient E.coli cells will heighten oxidization of disulphide bonds ( Basette et al, 1999 ) . In a old survey by Peisley and Goole ( 2007 ) , the FNII sphere of MMP-2 was successfully expressed in BL21trxB ( DE3 ) cells, the mutant strain of BL21 which is deficient in thioredoxin reductase, as recombinant proteins with a Trx-tag. There are besides assorted illustrations where the usage of thioredoxin merger protein in BL21trxB ( DE3 ) were successful in the look of soluble proteins with multiple disulphide bonds ( Wilkinson et al, 2004 ; Jurado et Al, 2006 ) .
In this experiment, the FXII-RFPs were expressed in BL21 ( DE3 ) cells, non the mutation strain which is deficient in thioredoxin reductase. Yet, as mentioned above, the formation of disulphide bonds were successful. There are besides many other surveies where the usage of Trx-fusion ticket in BL21 ( DE3 ) have successfully expressed the merger proteins as soluble proteins ( Ribas et al, 2000 ; Simmons et Al, 2006 ; Wang et Al, 2008 ) . So how did Trx-tag mediate oxidization of disulphide bonds in the presence of thioredoxin reductase in BL21 ( DE3 ) ? Trx has been demonstrated to help protein turn uping in a mechanism that is independent of their oxidation-reduction activity in a survey by Berndt et Al ( 2008 ) . In their survey, Trx with mutants at the ‘active site ‘ which is of import for contact action of oxidative protein folding was every bit efficient as the wild type Trx merger spouse in the initiation of right folding. It has been suggested that Trx which is fused to a protein of involvement may move as molecular chaperone to forestall the precipitation and collection of the amalgamate nascent spouse proteins until they have reach a stable folding province. Trx is a compact, extremely soluble protein with robust turn uping features ( LaVallie et al, 2000 ) . Its high solubility may forestall the collection of the amalgamate proteins while its ability to make native conformation quickly may advance the downstream merger spouse to follow their right construction every bit good.
4.3 Successful HIS-TRX TAG CLEAVAGE USING 3C PROTEASE
Although MALDI-TOF can find protein mass with an truth of at least 0.1 per 1000, it works best with proteins from 30-40kDa ( Rehm, 2006 ) . The MW of His-Trx ticket, FXII1-71 and FXII23-71 are about 13.8kDa, 8.45kDa and 5.86kDa severally. Peptide fragments generated from these proteins with such low MW will non be sufficient to fit against protein databases which explain why consequences obtained with MALDI-TOF analysis in this survey were inconclusive.
For LC-MS/MS analysis, it is of import to observe that non all fragment ions produced during MS can be present at a degree which is noticeable ; therefore several peptides from the mark protein should let us to place the mark protein with comparative certainty. It was suggested that a stretch of seven aminic acids is frequently alone to a specific protein in the human genome ( Rosenberg, 2004 ) .
4.3.1 Analysis of Tag Cleavage from f-FXII1-71 and f-FXII23-71
22.214.171.124 Designation of His-Trx Tag
In this survey, LC-MS/MS analysis of the in-gel digestion merchandises of set 1 and 3 ( Figure 4.3 ) identified at least four ion multitudes that matched the sequence of E.coli Trx, with each of the fragments holding more than seven aminic acids. In add-on, none of the peptide fragments were matched to FXII protein of any species. These confirm that the Trx-tag has been successfully cleaved utilizing 3C peptidase.
Figure 4.3: The 18 % SDS-PAGE gel ( from Figure 3.4B ) used to analyze the proteolytic ticket remotion by 3C peptidase.
126.96.36.199 Analysis of His-Trx ticket Cleavage from f-FXII1-71
De novo peptide sequencing identified three integral fragment ion multitudes from set 4 that matched that of FXII with each fragment dwelling of a stretch of at least 13 aminic acids. Furthermore, 65 of the 71 aminic acids of FXII1-71 were identified which suggest that the whole FXII1-71 protein is integral in set 4. Based on these consequences, we can confidently reason that set 4 corresponds to FXII1-71 and confirm that the active site of 3C peptidase is located between His-Trx ticket and FXII1-71 as demonstrated by Walker et Al ( 1994 ) .
Yet, two peptide ion multitudes that were identified to Trx were besides found in in-gel digestion merchandises of set 4. It is improbable that this is a consequence of uncomplete ticket cleavage from f-FXII1-71 because f-FXII1-71 has a higher MW ( ~22kDa ) and therefore should be located higher up in the SDS-PAGE gel. These two fragment ion multitudes generated in set 4 were indistinguishable to those found in set 3. Note that the in-gel digestion merchandises of set 4 were analysed after set 3. It was besides found that the peptide ion multitudes of set 3 produced really high signals which besides did look during the clean tally between tallies of set 3 and 4 during LC-MS/MS. Therefore, there is a possibility that the Trx signals identified in set 4 were taints carried-over from the tally of set 3. On top of that, the presence of Trx fragments in set 4 could be due to the ill resolved set 3 and 4 on the 18 % SDS-PAGE gel. As seen in Figure 4.3, set 3 and band 4 were located really near to each other and there is non a distinguishable boundary line between the two sets. Therefore, there is a possibility that when the sets were incised from the gel, a little part of set 3 remained integral in set 4. To farther elucidate this, we can reiterate the proteolysis test and utilize other fresh 2D gel techniques which have better separation declaration. This will be discussed in more item in subdivision 4.3.2.
188.8.131.52 Analysis of His-Trx Tag Cleavage from f-FXII23-71
De novo peptide sequencing identified two integral peptide fragments in set 2 that correspond to FXII which consist of more than seven back-to-back amino acids and covered 34 of the 49 aminic acids of FXII23-71. Unlike set 4, Trx was non detected in set 2 which suggests that all the merger protein f-FXII23-71 have been cleaved go forthing merely the FXII23-71. This farther suggests that the hints of Trx detected in set 4 were due to taints instead than uncleaved merger protein. At this point of the treatment, it can be confirmed that 3C peptidase can be used to take the His-Trx ticket from f-FXII1-71 and f-FXII23-71.
184.108.40.206 Stability of FXII-RPs after ticket remotion
In the 18 % SDS-PAGE gel used for analysis of tag cleavage ( Figure 4.3 ) , the sets that correspond to FXII23-71 and FXII1-71 ( band 2 and 4 ) have lower strengths than the sets that correspond to His-Trx ticket ( band 1 and 3 ) . The rule of gel staining utilizing coomassie dye is that the dye will adhere to groups of aminic acids. As the FXII-RPs have lower MWs than the His-Trx ticket, of course less dye will adhere to them as compared to His-Trx ticket. Therefore, it is non unusual for the FXII-RPs to be stained less intensely than the His-Trx ticket.
However, this difference in strength may be contributed by another factor. In this survey, although we have proven that the Trx has aided disulphide bond formation, we do non cognize whether the disulphide bonds formed were lasting, i.e. will the FXII recombinant proteins still remain soluble upon remotion of the His-Trx ticket. It is of import to observe that there were instances where one time the merger spouse was removed, the amalgamate recombinant protein does non stay soluble ( Waugh, 2005 ; Esposito and Chatterjee, 2006 ) . A hypothesised account for this observation is that in the presence of the solubility-enhancing ticket, the apparently soluble amalgamate proteins are held in solution as ‘soluble sums ‘ by interactions with the merger spouse ( Esposito and Chatterjee, 2006 ) . Thus, one time the solubility-enhancing ticket is removed, the amalgamate proteins return to their aggregative province.
If the FXII-RPs did act this manner, they will precipitate out of the solution after tag cleavage ensuing in their lower concentration in the solution as compared to the His-Trx ticket ; accordingly, when analysed on SDS-PAGE gel, they will be less intensely stained. As the volume of reaction mixture used in this survey is really little, it is non possible to detect any precipitates if any were formed. To verify this, we can scale up this test utilizing more FXII-RFPs and peptidase to give a larger reaction volume so that it can be observed whether any precipitates are being formed. In add-on, after separation of the His-Trx ticket and FXII merger proteins via GFC, these proteins can be analysed quantitatively to find their concentration and later compare their comparative output.
4.3.2 Analysis of Tag Cleavage from f-FXII23-112
On the 18 % SDS-PAGE gel ( Figure 4.4 ) , we can see that there is merely one seeable set in the lanes ( 3, 7, 10 ) which contain cleavage merchandises of f-FXII23-112. These sets are on the same horizontal line as the His-Trx tickets whose individuality have been proved in MS analysis as mentioned above. This proved that the His-Trx ticket was cleaved from f-FXII23-112 but it is instead uneven that there are no other sets seen in these lanes because His-Trx ticket cleavage from f-FXII23-112 should give both His-Trx and FXII23-112. An account for this observation is that the FXII23-112 was retained in the same set as His-Trx in the gel. This suggestion is based on the hapless separation of the sets seen in lane 8. The proteins in these sets have been identified as His-Trx ( ~13.8kDa ) and FXII1-71 ( ~8.45kDa ) severally via MS. In lane 8, we can see that the set which corresponds to FXII1-71 is located merely below the His-Trx set. If a protein of ~8.45kDa MW is so ill resolved from a protein of ~13.8kDa MW in the gel, it is improbable that FXII23-112, which has a MW of ~10.27kDa, will organize a distinguishable set on the gel.
Figure 4.4: The 18 % SDS-PAGE gel ( from Figure 3.4B ) used for analysis of His-Trx ticket cleavage. Lane 3, 7 and 10 contains proteolytic merchandises of 4hr, 6hr and nightlong incubation of f-FXII23-112 with 3C peptidase.
To clarify this, farther experiments can be done utilizing separation techniques with better deciding power in order to divide the FXII23-112 and His-Trx ticket clearly. For better separation, a 20 % SDS-PAGE gel can be used. Alternatively, we can utilize the Tricine gel system which a has high deciding power in the 5-20kDa scope ( Sch & A ; auml ; gger and von Jagow, 1987 ) .
4.4 CRYSTALLISATION SCREEN YIELDED MICRO-CRYSTALS
4.4.1 Designation of Protein Crystals
Precipitates were observed in the crystallization status 0.1 M Na ethanoate pH 4.6 and 1.0 M Na chloride for f-FXII1-71 and their double refraction belongingss confirmed that these are crystals. It is of import to observe that besides protein crystals, many other organic and inorganic stuffs that may be present within crystallization screen are birefringent every bit good, salt crystals being the most common one. Therefore, it is of import to place protein crystals from others. There are several ways to distinguish a protein crystal from a salt one. Typically, the double refraction belongingss of salt crystals are more marked than that of protein crystals ( Bergfors, 1999 ) . Besides this, salt crystals tend to hold crisp borders and are larger in size. They are besides able to turn within a short period of clip. The crystals as seen in Figure 3.8B were merely somewhat birefringent and comparatively little. Furthermore, they started to turn merely after three hebdomads upon puting up the screen. All these strongly suggest that these are proteins crystals instead than salt. Although there are assorted other trials which can more decidedly turn out that these are protein crystals, these trials are unwanted as they involve break of the crystals.
4.4.2 Has Trx Aided Crystallisation?
The reservoir solution in which crystals of f-FXII1-71 were formed has a pH of 4.6. Ideally, the pH of the reservoir solution should be as near to the protein ‘s isolectric point ( IEP ) as possible because protein carries a net charge of nothing at its IEP. However, the estimated IEP of f-FXII1-71 is 6.09. Interestingly, the estimated IEP of the thioredoxin sequence has an IEP of 4.8 which is reasonably similar to the pH of the reservoir. The IEP of FXII1-71 on the other manus was found to be 7.06. There are several studies where Trx-tag was demonstrated to help crystallization of its merger spouse either by moving as a bearer or a stabilizing agent. In the former, the Trx-tag remained fused to the spouse protein ( Stoll et al, 1998 ; Corsini et Al, 2008 ) whereas in the latter the ticket was cleaved but non removed ( Cura et al, 2007 ) . Therefore, there is a possibility that in this survey, the crystallization status is suited for the crystallization of Trx and that its crystallization has aided the FXII-RPs to clear every bit good.
4.4.3 Optimization of Crystallisation Screen
In Figure 3.8, we can see that overlapping acerate micro-crystals were formed from two nucleation sites. These crystals are non ideal for analysis as they are excessively little to be characterised by diffraction ( Bodenstaff et al, 2002 ) . Besides, single crystals are required for X-ray diffraction analysis ( Judge et al, 2005 ) .
The crystallization procedure can be divided into two stages: nucleation and crystal growing. Ironically, the conditions required for these two stages are frequently different. For nucleation, a supersaturated solution is indispensable whereas growing tend to happen in a solution where the concentration is merely somewhat below that of the supersaturated. In the crystallization stage diagram, this is known as the metabstable zone which lies between supersaturation and solubility. The metastable zone supports crystal growing but no farther nucleation will happen. Therefore, to obtain good quality well-ordered crystals, it is frequently necessary to divide nucleation from growing. One of the techniques which can be used to accomplish this is seeding where crystals nuclei formed are introduced straight into a metastable protein solution ( Chayen, 2005 ; Benvenuti and Mangani, 2007 ) .
Crystallization is normally the rate-limiting in protein construction finding via X-ray crystallography as there are a big figure of variable factors that can impact crystals growing ( Benvenuti and Mangani, 2007 ) . Important parametric quantities that may act upon crystal formation include temperature, protein concentration, pH, buffer type, ionic strength. To increase success rate, one must invariably measure the feedback obtained from each screen ( Bodenstaff et al, 2003 ; Benvenuti and Mangani, 2007 ) . Now that we know the conditions that support nucleation of f-FXII1-71, this is a good starting point for subsequent optimization showing.
4.5 STRUCTURAL-BASED FUNCTIONAL ANALYSIS OF FNII
4.5.1 The Rationale of Functional Annotations Based on Sequence Homology
Through development, functionally and structurally of import residues tend to be more conserved as compared to other places in the sequence, therefore sequence homology has been suggested to be a utile tool in functional assignment of proteins ( Goldsmith-Fischman and Honig, 2003 ; Petsko and Ringe, 2004 ) . A 40 % regulation ( Figure 4.5 ) has been put frontward based on the analysis of the biochemical map of proteins with sequence similarity ; if a protein portions more than 40 % sequence individuality with a mark protein whose biochemical map is known, it is sensible to presume that the two proteins may hold a common biochemical map with the status that the functionally of import residues are conserved between the two sequences ( Petsko and Ringe, 2004 ) .
Figure 4.5: The relationship between sequence similarity and functional similarity. When the sequence individuality of two proteins is above 40 % , it is really likely that these two proteins have similar biochemical map. This graph is obtained by Mark Gerstein by plotting the per centum of proteins brace with the exact biochemical map against the per centum of sequence individuality ( Petsko and Ringe, 2004 ) . ( Adapted from hypertext transfer protocol: //bioinfo.mbb.yale.edu/lectures/spring2002/show/pages/Slide16.htm )
4.5.2 Multiple Sequence Alignment of FNII sphere
Sequence alliance of FXII FNII sequence to that of fibronectin and MMP-9 both show sequence similarity of more than 40 % ( Figure 4.6 ) , therefore fibronectin and MMP-9 are suited marks for foretelling the map of FXII FNII sphere. The FNII spheres of both fibronectin and MMP-9 have been implicated in adhering to collagen ( Pickford et al, 1997 ; Visse and Nagase, 2003 ) .
In a survey of the solution construction of the first type II faculty of fibronectin, it was demonstrated that the type II faculty is involved in adhering to collagen and that hydrophobic interaction is of import. It was proposed that in this FNII sphere, the big solvent-accessible hydrophobic pocket formed by the residues, Tyr21, Trp40, Tyr47, Tyr53 and Phe55, is responsible for collagen binding ( Pickford et al, 1997 ) . Multiple sequences alliance ( Figure 4.6 ) shows that the places in the FNII sequence of FXII and MMP-9 that correspond to the places of Tyr21, Trp40, Tyr47, Tyr53 and Phe55 in fibronectin consist of hydrophobic aromatic residues tyrosine, tryptophan or phenylalanine.
Using the homology theoretical account of FXII FNII sphere, it was found that these conserved hydrophobic places do organize a dissolver accessible pocket every bit good ( Figure 4.7A ) . Furthermore, the surface around this pocket has got a net positively charged charge as shown in Figure 4.7B. Besides, the side concatenation of arginine which is positively-charged is located near to the hydrophobic pocket and this arginine residue is conserved in other FNII spheres every bit good.
FN 1st nlngepcvlpftyngrtfyscttegrqdghlwcsttsnyeqdqkysfc
Figure 4.6: The multiple sequences alliance of the first FNII sphere of fibronectin ( FN 1st ) , FNII sphere of FXII and the first FNII sphere of MMP-9. The five residues highlighted ( cyan ) in the FNII sphere of fibronectin ( Tyr21, Trp40, Tyr47, Tyr53 and Phe55 ) form a hydrophobic pocket which is indicated in collagen binding. Hydrophobic residues ( ruddy ) were found to be conserved in the places of FXII and MMP-9 sequences that correspond to Tyr21, Trp40, Tyr47, Tyr53 and Phe55. The conserved arginine residues that are located near to the hydrophobic residues are highlighted ( green ) .
( A )
( B )
Figure 4.7: A thread representation of the FNII sphere of FXII. The residues in ruddy are the conserved hydrophobic residues. The side-chain of arginine ( purple ) which is positively-charged is located near to the pocket. ( A ) The dissolver accessible surface is generated and the hydrophobic pocket is found to be accessible to solvent ; ( B ) The electrostatic surface is generated and the hydrophobic pocket is found to be located near to a net positively charged surface.
As mentioned before, the sequence consisting of aminic acids 39-47, which lies within the FNII sphere of FXII, is involved in adhering to negatively-charged surfaces ( Citarella et al, 2000 ) . Besides, it was demonstrated in assorted literatures that FXII activation is initiated upon adhering to negatively-charged surfaces ( Cochrane et al, 1973 ; Griffin, 1978 ; Samuel et Al, 1992 ) . The positively charged surface and the positively-charged side-chain fulfil the features of a binding site for negatively-charged surfaces. As collagen ( negatively-charged ) is besides known to trip FXII, based on the high sequence individuality of the FNII sphere of FXII to that of fibronectin and MMP-9, there is a possibility that the conserved hydrophobic pocket seen in Figure 4.7 could be the putative binding site for negatively-charged surfaces.
Yet, this is merely an premise as there are many unknown variables. For illustration, we do non cognize if this hydrophobic pocket is accessible within the whole FXII construction as the pocket may be sterically hindered by other spheres ; the conformation or the open residues of the FNII sphere could be different in world when this sphere exists as portion of the FXII concatenation. In add-on, the precise mechanism of FXII activation is non known yet.
In decision, we have successfully expressed the FXII-RFPs, f-FXII1-71, f-FXII23-71, f-FXII23-112 and f-FXII23-154, in BL21 ( DE3 ) cells with His-Trx ticket at the N-terminus. These were found to be soluble. Several analyses have proved that f-FXII1-71 and f-FXII23-71 have adopted the right globular conformation which suggests that the Trx merger ticket has aided disulphide bonds formation. We believe that this would besides be the instance for f-FXII23-112 and f-FXII23-154 if clip has allowed for their farther word pictures. Step-wise purifications of the FXII-RFPs via IMAC and GFC resulted in extremely homogenous sample. A crystallization screen for f-FXII1-71 has produced crystals which we believe are protein crystals. The pH status of the reservoir was found to be really near to the IEP of Trx proposing the possibility that the Trx might hold an assisting function in the crystallization of f-FXII1-71. A test to split the His-Trx ticket in f-FXII1-71 and f-FXII23-71 utilizing 3C peptidase was successful but farther probes are required to find the stableness of the FXII-RPs after tag cleavage. Homology theoretical account of the FNII sphere of FXII shows the being of a conserved hydrophobic pocket that is solvent-accessible ; this hydrophobic pocket may be functionally important.
4.7 FUTURE WORKS
Due to clip restraint, there were many things that we did non pull off to formalize in this survey. Now that we know the E.coli look system used is suited for the look of FXII-RFPs, the following measure would be to scale up the production of these merger proteins. Further word pictures of these merger proteins via IMAC/GFC may uncover more information on their third construction. In order to clarify the function that Trx has in the solubility of the merger proteins, the cleavage of His-Trx ticket should be repeated at a larger graduated table to find if the FXII proteins still remain soluble in the absence of Trx. Besides, more crystallization screens of the proteins with/without ticket can be set up to obtain a good quality crystal for X-ray diffraction analysis. As this survey is portion of an ongoing undertaking, large-scale production of f-FXII1-71 is traveling on as the crystallization screen in this survey demonstrated that f-FXII1-71 has successfully yielded protein crystals. If good quality crystals can be obtained, diffraction analysis will uncover the third construction the FNII sphere.