**3. Unfolding/refolding of** *Rhipicephalus annulatus* **GST mu class**

GSTs are dimeric proteins composed of identical or structurally related subunits. Each subu‐ nit has a molecular weight of about 25 kDa and is built of two domains and contains a com‐ plete active site consisting of a highly conserved G-site (GSH binding site) and a divergent H-site (Hydrophobic substrate binding site). The functional soluble enzymatic forms are found in dimers and only subunits within the same class can form heterodimers as found in alpha subunits, but this would not happen with either pi or mu subunits.

The nature of protein folding mechanisms and the manner in which the compact native state is achieved are still not well understood. From a wide range of experiments, it is now evi‐ dent that specific pathways of folding are involved, at least for many proteins. At equilibri‐

um, most monomeric and many oligomeric proteins display essentially a two-state pathway upon folding/unfolding, for which thermodynamically stable folding intermediates do not exist. Other mechanisms result in the formation of stable intermediates. These monomeric intermediates sometimes have preserved tertiary structure or appear as molten globules [18-20]. For proteins composed of subunits, the intermediates are either partially folded oli‐ gomeric states or monomeric states.

R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to *Rhipicephalus appendiculatus* GST (AAQ74442). The conservation scoring was performed by PRALINE software (http://zeus.few.vu.nl). The scoring scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position.

**H. long. M A - - P I L G Y W D I R G L A Q P I R L L L A H A D V K V E D K R Y S C G P P P D F D R S A W L K R. annulatus M A - - P V L G Y W D I R G L A Q P I R L L L A H V D A K V D D K R Y S C G P P P D F D R S S W L N R. micro 2 M A - - P V L G Y W D I R G L A Q P I R L L L A H V D A K V D D K R Y T C G P P P D F D R S S W L N R. micro (I) M A - - P V L G Y W D I R R L A Q P I R L L L A H V D A K V D D K R Y S C G P P P D F D R S S W L N R. append. M A - - P I L G Y W N I R G L A Q P F R L L L A H V D A K V E E K Q Y S C G P P P D F D K S Y W L S R. micro 1 M A P T P V V G Y T T A R G L A Q S I R N L L V Y K G V H F E D K R Y E F G P A P T Y E K L G W A A**

**H. long. E K H T L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L D G K T E A E K Q R V D R. annulatus E K T K L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. micro 2 E K T K L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. micro (I) E K T K L G L E F P N L P Y Y I D S D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. append. E K P K L G L D F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K Y D L M G K T E A E K Q R V D R. micro 1 D S A S L G F T F P N L P Y Y I D G D V R L T Q S L A I L R Y L G K K H G L D A R S D Q E A A E L W**

**H. long. V T E Q Q F A D F R M N W V R M C Y N P D F D K L K V D Y L K N L P D A L K S F S E Y L G K H K F F R. annulatus V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. micro 2 V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. micro (I) V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. append. V V E Q Q L A D F R V N W G R L C Y S P D F E K L K G D Y L K D L P A S L K A F S D Y L G N R K F F R. micro 1 L M E Q Q A N D L L W A L V V T A M N P N A T E A R K S Q E K R L A D S L P R W Q E L L K K R R W A**

**H. long. A G D H V T Y V D F I A Y E M L A Q H L L L A P D C L K D F P N L K A F V D R I E A L P H V A A Y L R. annulatus A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. micro 2 A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. micro (I) A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. append. A G D N L T Y V D F I A Y E M L D Q H L L F A P D C L K D F A N L K A F V D R V A A L P R V A A Y L R. micro 1 L G N T L T Y V D F L L Y E A L D W N R Q F A P D A F A N R P E L L D Y L R R F E Q L P N L K E Y F**

 **Figure 2.** Three state (H, E C) secondary structure of Glutathione S-transferase mu class sequences from different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to *Rhipicephalus appendiculatus* GST (AAQ74442). The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no

**Figure 2.** Three state (H, E C) secondary structure of Glutathione S-transferase mu class sequences from different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to Rhipi‐ *cephalus appendiculatus* GST (AAQ74442). The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no DSSP information available, or that no pre‐

Eftink et al. [18] proposed three models for proteins unfolding; the first is the two-state mod‐ el. This model assumes that the protein exists only as a native dimer, D, and unfolded mon‐ omers, U, [D←→ 2U]. The second is three-state model (Folded monomers model). Unlike the first model, this one assumes that there is a two-step (three-state) unfolding process, with the formation of a folded monomeric intermediate, N. "folded" means that the inter‐ mediate can be further unfolded, in a cooperative manner, by addition of denaturant. How‐ ever, it is not known to what extent the intermediate's structure actually resembles the

**. . . . . . . . . 210 . . . . . . . . . 220 . . . . .**

10 DSSP information available, or that no prediction was possible for that sequence.

**H. long. K S D K C I S W P L N G D M A S F G S R L Q K K P R. annulatus K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro 2 K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro (I) K S D K C I K W P L N G D M A S F G S R L Q K K P R. append. K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro 1 A S D K Y V K W P I M A P Y M F W G H K - - - - -**

**. . . . . . . . . 10 . . . . . . . . . 20 . . . . . . . . . 30 . . . . . . . . . 40 . . . . . . . . . 50** 

**. . . . . . . . . 60 . . . . . . . . . 70 . . . . . . . . . 80 . . . . . . . . . 90 . . . . . . . . . 100**

**. . . . . . . . . 110 . . . . . . . . . 120 . . . . . . . . . 130 . . . ...... 140 . . . . . . . . . 150**

**. . . . . . . . . 160 . . . . . . . . . 170 . . . . . . . . . 180 . . . ...... 190 . . . . . . . . . 200**

5

271

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482

Running Title

1

2

11

diction was possible for that sequence.

**Unconserved** 0 1 2 3 4 5 6 7 8 9 10 **Conserved**

4 Book Title


4 long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is the *Rhipicephalus*  5 *annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. 6 micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), R. micro 1 refers to 7 *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to *Rhipicephalus appendiculatus* **Figure 1.** Amino acid conservation of Glutathione S-transferase mu class of different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is the *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064),

3 **Figure 1.** Amino acid conservation of Glutathione S-transferase mu class of different tick species. H.

8 GST (AAQ74442). The conservation scoring was performed by PRALINE software

1

2

R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to *Rhipicephalus appendiculatus* GST (AAQ74442). The conservation scoring was performed by PRALINE software (http://zeus.few.vu.nl). The scoring scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position. Running Title 5

**Unconserved** 0 1 2 3 4 5 6 7 8 9 10 **Conserved**

1

2

um, most monomeric and many oligomeric proteins display essentially a two-state pathway upon folding/unfolding, for which thermodynamically stable folding intermediates do not exist. Other mechanisms result in the formation of stable intermediates. These monomeric intermediates sometimes have preserved tertiary structure or appear as molten globules [18-20]. For proteins composed of subunits, the intermediates are either partially folded oli‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

4 Book Title

**H. long. M A - - P I L G Y W D I R G L A Q P I R L L L A H A D V K V E D K R Y S C G P P P D F D R S A W L K R. annulatus M A - - P** V **L G Y W D I R G L A Q P I R L L L A H V D A K V D D K R Y S C G P P P D F D R S S W L N R. micro 2 M A - - P** V **L G Y W D I R G L A Q P I R L L L A H V D A K V D D K R Y T C G P P P D F D R S S W L N R. micro (I) M A - - P** V **L G Y W D I R R L A Q P I R L L L A H V D A K V D D K R Y S C G P P P D F D R S S W L N R. append. M A - - P I L G Y W N I R G L A Q P F R L L L A H V D A K V E E K Q Y S C G P P P D F D K S Y W L S R. micro 1 M A P T P V V G Y T T A R G L A Q S I R N L L V Y K G V H F E D K R Y E F G P A P T Y E K L G W A A Consistency \* \* 0 0 \* 9 8 \* \* 7 6 7 \* 7 \* \* \* 7 8 \* 7 \* \* 8 8 6 7 7 7 7 7 8 \* 8 \* 7 7 \* \* 7 \* 7 8 8 8 7 5 \* 7 5**

**H. long. E K H T L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L D G K T E A E K Q R V D R. annulatus E K T K L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. micro 2 E K T K L G L E F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. micro (I) E K T K L G L E F P N L P Y Y I D S D V K L T Q S M A I L R Y L A R K H G L E G K T E A E K Q R V D R. append. E K P K L G L D F P N L P Y Y I D G D V K L T Q S M A I L R Y L A R K Y D L M G K T E A E K Q R V D R. micro 1 D S A S L G F T F P N L P Y Y I D G D V R L T Q S L A I L R Y L G K K H G L D A R S D Q E A A E L W Consistency 8 8 4 6 \* \* 8 6 \* \* \* \* \* \* \* \* \* 8 \* \* 8 \* \* \* \* 9 \* \* \* \* \* \* 8 8 \* 8 7 \* 5 8 8 8 8 7 \* 7 7 8 8 6**

**H. long. V T E Q Q F A D F R M N W V R M C Y N P D F D K L K V D Y L K N L P D A L K S F S E Y L G K H K F F R. annulatus V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. micro 2 V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. micro (I) V S E Q Q F A D F R M N W V R L C Y N P D F E K L K G D Y L K N L P A S L K A F S D Y L G T H K F F R. append. V V E Q Q L A D F R V N W G R L C Y S P D F E K L K G D Y L K D L P A S L K A F S D Y L G N R K F F R. micro 1 L M E Q Q A N D L L W A L V V T A M N P N A T E A R K S Q E K R L A D S L P R W Q E L L K K R R W A Consistency 8 5 \* \* \* 6 7 \* 8 7 6 7 7 7 7 7 7 7 8 \* 8 7 6 8 7 8 5 8 7 7 \* 6 \* 7 5 8 \* 7 6 8 8 8 7 \* 7 5 6 8 8 7**

**H. long. A G D H V T Y V D F I A Y E M L A Q H L L L A P D C L K D F P N L K A F V D R I E A L P H V A A Y L R. annulatus A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. micro 2 A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. micro (I) A G D N L T Y V D F I A Y E M L A Q H L I F A P D C L K D F A N L K A F V D R I E A L P H V A A Y L R. append. A G D N L T Y V D F I A Y E M L D Q H L L F A P D C L K D F A N L K A F V D R V A A L P R V A A Y L R. micro 1 L G N T L T Y V D F L L Y E A L D W N R Q F A P D A F A N R P E L L D Y L R R F E Q L P N L K E Y F Consistency 7 \* 8 6 8 \* \* \* \* \* 9 7 \* \* 7 \* 5 7 8 7 6 8 \* \* \* 7 8 7 8 7 6 8 \* 7 7 8 8 7 \* 7 7 7 \* \* 6 8 7 7 \* 8**

 **Figure 1.** Amino acid conservation of Glutathione S-transferase mu class of different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is the *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to *Rhipicephalus appendiculatus* GST (AAQ74442). The conservation scoring was performed by PRALINE software

**Figure 1.** Amino acid conservation of Glutathione S-transferase mu class of different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is the *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064),

**. . . . . . . . . 210 . . . . . . . . . 220 . . . . .**

**H. long. K S D K C I S W P L N G D M A S F G S R L Q K K P R. annulatus K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro 2 K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro (I) K S D K C I K W P L N G D M A S F G S R L Q K K P R. append. K S D K C I K W P L N G D M A S F G S R L Q K K P R. micro 1 A S D K Y V K W P I M A P Y M F W G H K - - - - - Consistency 7 \* \* \* 7 9 8 \* \* 9 7 8 7 7 7 7 8 \* 7 8 6 6 6 6 6**

**Unconserved** 0 1 2 3 4 5 6 7 8 9 10 **Conserved**

1

2

**. . . . . . . . . 10 . . . . . . . . . 20 . . . . . . . . . 30 . . . . . . . . . 40 . . . . . . . . . 50**

**. . . . . . . . . 60 . . . . . . . . . 70 . . . . . . . . . 80 . . . . . . . . . 90 . . . . . . . . . 100**

**. . . . . . . . . 110 . . . . . . . . . 120 . . . . . . . . . 130 . . . . . . . . . 140 . . . . . . . . . 150**

**. . . . . . . . . 160 . . . . . . . . . 170 . . . . . . . . . 180 . . . . . . . . . 190 . . . . . . . . . 200**

gomeric states or monomeric states.

Applications

270

3 **Figure 2.** Three state (H, E C) secondary structure of Glutathione S-transferase mu class sequences 4 from different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. 5 annulatus is *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus*  6 *microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), 7 R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to 8 *Rhipicephalus appendiculatus* GST (AAQ74442). The Helix (H) structure is in red and the Strand (E) 9 is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no 10 DSSP information available, or that no prediction was possible for that sequence. **Figure 2.** Three state (H, E C) secondary structure of Glutathione S-transferase mu class sequences from different tick species. H. long*.* refers to *Haemaphysalis longicornis* GST (AAQ74441), R. annulatus is *Rhipicephalus annulatus* GST (ABR24785), R. micro 2 refers to *Rhipicephalus microplus* GST (AAD15991), R. micro I is *Rhipicephalus microplus* Indian strain GST (ADQ01064), R. micro 1 refers to *Rhipicephalus microplus* GST (AF366931\_1), and R. append. refers to Rhipi‐ *cephalus appendiculatus* GST (AAQ74442). The Helix (H) structure is in red and the Strand (E) is in blue. The sequence in the alignment has no color assigned for the coil (C) because there is no DSSP information available, or that no pre‐ diction was possible for that sequence.

11 Eftink et al. [18] proposed three models for proteins unfolding; the first is the two-state mod‐ el. This model assumes that the protein exists only as a native dimer, D, and unfolded mon‐ omers, U, [D←→ 2U]. The second is three-state model (Folded monomers model). Unlike the first model, this one assumes that there is a two-step (three-state) unfolding process, with the formation of a folded monomeric intermediate, N. "folded" means that the inter‐ mediate can be further unfolded, in a cooperative manner, by addition of denaturant. How‐ ever, it is not known to what extent the intermediate's structure actually resembles the

subunits of the native dimer [D←→2N←→2U]. The third model is also three-state model and also considers the existence of an intermediate in the unfolding process but the inter‐ mediate is a partially unfolded dimeric state, D', which can then be further unfolded to un‐ folded monomers [D←→ D'←→2U].

[22, 23]. Clear evidence of acidic pH induced stable folding intermediates has been obtained with some lipocalins, such as β-lactoglobulin [24, 25], retinol binding protein [26] and

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482 273

Electrostatic interactions between charged residues on the surface of a protein play an im‐ portant role in conferring stability to its folded structure. Change of pH alters the ionization state of these residues, causing intramolecular charge repulsion and possible disruption of salt bridges that can lead to destabilization of the native protein conformation [28]. pH is an important factor determining protein structure and function. Most proteins are stable and active at physiological pH and show varying degrees of denaturation in acid medium. How‐ ever, as the acid concentration increases, a number of these proteins revert back to a com‐ pact conformation containing substantial secondary structure that resemble the folding intermediates known as molten globules [29, 30]. Study of the structural stability of a pro‐ tein as a function of pH thus helps understand the thermodynamic or kinetic intermediates in its folding pathway and identifies the electrostatic interactions important for the stability

In ticks, no data is available about the unfolding pathway of GST classes except for the *Rhi‐ picephalus* (*Boophilus*) *annulatus* (*R. annulatus*) recombinant GST mu class (BaGSTM) [33]. Be‐ cause of the non-identity of the different transitions monitored, the acid denaturation of BaGSTM does not appear to be a simple two-step transition, rather a multi-step process dur‐

Shahein et al. [34] cloned the GST mu class from λZAP cDNA library of *R. annulatus*. The GST protein (Figure 3) contains four tryptophan residues (Try 7, Try 45, Try 110 and Try 214) and ten tyrosine residues [34]. Comparison of BaGST with the protein databank for GST sequences revealed the presence of the SMAILRYL motif that may play an important struc‐ tural role in GSH binding site and the interface domain The authors showed that the *E. coli* expressed recombinant protein (BaGSTM) exhibited peroxidatic activity on cumene hydro‐ peroxide sharing this property with GSTs belonging to the GST α class. The inhibition stud‐ ies using cibacron blue and bromosulfophthalein showed that the *R. annuatus* GST shares

In its native state, the BaGSTM enzyme exhibits an emission spectrum with a maximum at 329 nm (excitation 280 nm). This feature characteristic of tryptophan residues partially bur‐ ied in the protein matrix (Figure 4 a). The addition of increasing concentrations of GdmCl at equilibrium caused a red shift of λmax of the emission spectra from 329 nm to 352 nm. As shown in figure 4, compared with the native dimer, the fluorescence intensity increased with a slight red shift of λmax as the GdmCl concentration was increased. The intensity reached a maximum at approximately 1.45 M (partially unfolded dimer or nonnative dimer‐ ic intermediate). At GdmCl concentration between 1.5 M and 1.9 M there was no change in the fluorescence intensity or in λmax. The nonnative dimeric intermediate undergoes disso‐ ciation into monomeric intermediate at these GdmCl concentrations. Increasing the GdmCl concentration leads to another increase in the fluorescence intensity with a red shift of λmax and the intensity reached a maximum at approximately 2.4 M. This might be due to the for‐ mation of a partially unfolded monomer. After this concentration the intensity started to de‐

hGSTP1-1 [27].

of its folded state [31, 32].

ing which several intermediates coexist in equilibrium.

this property with the mammalian GST mu class.


**Table 1.** Glutathione S-transferase sequences identified in *Boophilus microplus* Gene Index (BmiGI). The asterisk means that there is no practical evidence of the subfamily. The assigned class is based on prediction similarity.

Full understanding of the protein folding process requires the identification and characteri‐ zation of all intermediate steps, which are often very transient and detected by kinetic stud‐ ies only. In these cases, some properties of the intermediates can be inferred, but little structural information can be derived from this approach. It is known that mild denaturing conditions, such as moderately high temperature or low pH, promote partially unfolded states that are similar to those observed at moderate concentrations of guanidinium chloride [19]. Therefore a large number of studies have been performed on these partially folded states [21]. Some of these more or less stable intermediates, called ''molten globules''are characterized by a largely conserved secondary structure but loss of tertiary structure and, due to the presence of a loosely packed hydrophobic core, binding of ANS is often observed [22, 23]. Clear evidence of acidic pH induced stable folding intermediates has been obtained with some lipocalins, such as β-lactoglobulin [24, 25], retinol binding protein [26] and hGSTP1-1 [27].

subunits of the native dimer [D←→2N←→2U]. The third model is also three-state model and also considers the existence of an intermediate in the unfolding process but the inter‐ mediate is a partially unfolded dimeric state, D', which can then be further unfolded to un‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

TC 298 908 *Homo sapiens* Omega 1 Omega 1 NP004823

TC2910 813 *R. microplus* Mu AAL99403

TC3737 825 *R. microplus* Mu AAD15991 TC1082 1236 *Mus musculus* Zeta 1 Zeta Q9WVL0 TC 811 859 *Anopheles gambiae* Zeta AAM61889 TC2689 1007 *Xenopus laevis* Mu CAD01094

**Table 1.** Glutathione S-transferase sequences identified in *Boophilus microplus* Gene Index (BmiGI). The asterisk means that there is no practical evidence of the subfamily. The assigned class is based on prediction similarity.

Full understanding of the protein folding process requires the identification and characteri‐ zation of all intermediate steps, which are often very transient and detected by kinetic stud‐ ies only. In these cases, some properties of the intermediates can be inferred, but little structural information can be derived from this approach. It is known that mild denaturing conditions, such as moderately high temperature or low pH, promote partially unfolded states that are similar to those observed at moderate concentrations of guanidinium chloride [19]. Therefore a large number of studies have been performed on these partially folded states [21]. Some of these more or less stable intermediates, called ''molten globules''are characterized by a largely conserved secondary structure but loss of tertiary structure and, due to the presence of a loosely packed hydrophobic core, binding of ANS is often observed

**(TC)-GST Class subfamily\***

*Galleria mellonella* Delta or Epsilon AAK64362

*Dermacentor variabilis* Delta or Epsilon AAO92279

**Accession number of the Hit**

**e value cutoff=0.001**

folded monomers [D←→ D'←→2U].

TC 213 861

Applications

272

TC2718 808

TC 614 756

TC3165 855 TC 762 1061 TC3881 831 TC4914 718 TC1038 742

TC3317 984

**Clone Length (bp) Top Blastx Hit**

Electrostatic interactions between charged residues on the surface of a protein play an im‐ portant role in conferring stability to its folded structure. Change of pH alters the ionization state of these residues, causing intramolecular charge repulsion and possible disruption of salt bridges that can lead to destabilization of the native protein conformation [28]. pH is an important factor determining protein structure and function. Most proteins are stable and active at physiological pH and show varying degrees of denaturation in acid medium. How‐ ever, as the acid concentration increases, a number of these proteins revert back to a com‐ pact conformation containing substantial secondary structure that resemble the folding intermediates known as molten globules [29, 30]. Study of the structural stability of a pro‐ tein as a function of pH thus helps understand the thermodynamic or kinetic intermediates in its folding pathway and identifies the electrostatic interactions important for the stability of its folded state [31, 32].

In ticks, no data is available about the unfolding pathway of GST classes except for the *Rhi‐ picephalus* (*Boophilus*) *annulatus* (*R. annulatus*) recombinant GST mu class (BaGSTM) [33]. Be‐ cause of the non-identity of the different transitions monitored, the acid denaturation of BaGSTM does not appear to be a simple two-step transition, rather a multi-step process dur‐ ing which several intermediates coexist in equilibrium.

Shahein et al. [34] cloned the GST mu class from λZAP cDNA library of *R. annulatus*. The GST protein (Figure 3) contains four tryptophan residues (Try 7, Try 45, Try 110 and Try 214) and ten tyrosine residues [34]. Comparison of BaGST with the protein databank for GST sequences revealed the presence of the SMAILRYL motif that may play an important struc‐ tural role in GSH binding site and the interface domain The authors showed that the *E. coli* expressed recombinant protein (BaGSTM) exhibited peroxidatic activity on cumene hydro‐ peroxide sharing this property with GSTs belonging to the GST α class. The inhibition stud‐ ies using cibacron blue and bromosulfophthalein showed that the *R. annuatus* GST shares this property with the mammalian GST mu class.

In its native state, the BaGSTM enzyme exhibits an emission spectrum with a maximum at 329 nm (excitation 280 nm). This feature characteristic of tryptophan residues partially bur‐ ied in the protein matrix (Figure 4 a). The addition of increasing concentrations of GdmCl at equilibrium caused a red shift of λmax of the emission spectra from 329 nm to 352 nm. As shown in figure 4, compared with the native dimer, the fluorescence intensity increased with a slight red shift of λmax as the GdmCl concentration was increased. The intensity reached a maximum at approximately 1.45 M (partially unfolded dimer or nonnative dimer‐ ic intermediate). At GdmCl concentration between 1.5 M and 1.9 M there was no change in the fluorescence intensity or in λmax. The nonnative dimeric intermediate undergoes disso‐ ciation into monomeric intermediate at these GdmCl concentrations. Increasing the GdmCl concentration leads to another increase in the fluorescence intensity with a red shift of λmax and the intensity reached a maximum at approximately 2.4 M. This might be due to the for‐ mation of a partially unfolded monomer. After this concentration the intensity started to de‐

crease with a red shift of λmax and at 4.0 M GdmCl, a λmax of 352 nm occurred, indicating the complete exposure of the tryptophan residues to the aqueous solvent which is consis‐ tence with the complete unfolding of the protein (unfolded monomer). From these results, at least two transition states between the native dimer and unfolded monomer could be identi‐ fied for BaGSTM.

10 Book Title

**Relative fluorescence intensity**

**b**

**d**

**Relative fluorescence intensity**

**0.8 0.9 1.0 1.1 1.2 1.3**

**F329/F352**

4 **Figure 4.** Fluorescence–emission spectra of BaGSTM (20 µg/ml) equilibrated in buffer A (20 mM 5 potassium phosphate buffer, pH 7.0 containing 1 mM EDTA/1 mM dithiothreitol) at 6 different GdmCl concentrations ranging from 0 to 4.0 M at room temperature. Excitation was done at

**Figure 4.** Fluorescence–emission spectra of BaGSTM (20 µg/ml) equilibrated in buffer A (20 mM potassium phosphate buffer, pH 7.0 containing 1 mM EDTA/1 mM dithiothreitol) at different GdmCl concentrations ranging from 0 to 4.0 M at room temperature. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm (a to e).

Unfolding was expressed as the ratio of fluorescence at 329 nm to the fluorescence at 352 nm (f).

**300 320 340 360 380 400**

**Wavelength (nm)**

**1.5 M to 1.9 M**

**None**

**1.4 <sup>f</sup>**

**300 320 340 360 380 400**

**Wavelength (nm)**

**0.0 0.5 1.0 1.5 2.0 2.5 3.0**

**[GdmCl]**

**1.0 M**

**None**

**1.2 M**

**0.7 M 0.8 M 0.9 M**

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482 275

**2.0 M**

**300 320 340 360 380 400**

**Wavelength (nm)**

**1.45 M**

**c (1.3, 1.35 and 1.4 M) 1.25 M**

**300 320 340 360 380 400**

**Wavelength (nm)**

**4.0 M 3.0 M 2.6 M 2.4 M**

**320 340 360 380 400**

**Wavelength (nm)**

**None**

**None**

**0.2 M 0.3 M 0.4 M 0.5 M 0.6 M**

**None**

**a**

**Relative fluorescence intensity**

**Relative fluorescence intensity**

**e**

**Relative fluorescence intensity**

1

2

3

**Figure 3.** Theoretical model of *R. annulatus* GST mu class (ABR24785) built with M4T server. The numbers of groups are 218, atoms 1765, and bonds 1813 [35, 36].

As shown in the figure 5, at the concentration of urea between 0 and 1.75 M, there was an increase in the fluorescence intensity, as the concentration of urea increased, without any de‐ tectable shift of λmax. The intensity of fluorescence was increased by 50% at 1.75 M urea concentration compared with the native state of the protein indicating a partial exposure of the fluorophore (first phase). Increasing the concentration of urea, between 2.0 M and 3.0 M, resulted in a slight red shift (by 3 nm) of the emission maximum (second phase). Whereas, the increase in fluorescence intensity decreased as the concentration of urea was increased. The fluorescence intensity at the end of the first phase was higher than that at the end of the second phase. This indicates the movement of the fluorophore back into a more hydropho‐ bic environment. At higher urea concentration, (between 3.25 and 4.5 M) the fluorescence in‐ tensity started to increase again with a shift of λmax (10 nm red shift). The fluorescence intensity was increased by three fold at 4.5 M urea compared to that of the native protein (third phase) [33].

Addition of higher concentrations of urea (5.0-7.0 M) did not change the intensity of the flu‐ orescence significantly compared to that at 4.5 M urea but progressively shifted the λmax to 347 nm. At 8.0 M urea, the fluorescence intensity was decreased again with a shift of λmax to 352 nm indicating the complete unfolding of the protein. The present results indicate that three intermediates could be identified between the native dimer and unfolded monomer during the unfolding of BaGSTM.

1

2

3

10 Book Title

crease with a red shift of λmax and at 4.0 M GdmCl, a λmax of 352 nm occurred, indicating the complete exposure of the tryptophan residues to the aqueous solvent which is consis‐ tence with the complete unfolding of the protein (unfolded monomer). From these results, at least two transition states between the native dimer and unfolded monomer could be identi‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 3.** Theoretical model of *R. annulatus* GST mu class (ABR24785) built with M4T server. The numbers of groups

As shown in the figure 5, at the concentration of urea between 0 and 1.75 M, there was an increase in the fluorescence intensity, as the concentration of urea increased, without any de‐ tectable shift of λmax. The intensity of fluorescence was increased by 50% at 1.75 M urea concentration compared with the native state of the protein indicating a partial exposure of the fluorophore (first phase). Increasing the concentration of urea, between 2.0 M and 3.0 M, resulted in a slight red shift (by 3 nm) of the emission maximum (second phase). Whereas, the increase in fluorescence intensity decreased as the concentration of urea was increased. The fluorescence intensity at the end of the first phase was higher than that at the end of the second phase. This indicates the movement of the fluorophore back into a more hydropho‐ bic environment. At higher urea concentration, (between 3.25 and 4.5 M) the fluorescence in‐ tensity started to increase again with a shift of λmax (10 nm red shift). The fluorescence intensity was increased by three fold at 4.5 M urea compared to that of the native protein

Addition of higher concentrations of urea (5.0-7.0 M) did not change the intensity of the flu‐ orescence significantly compared to that at 4.5 M urea but progressively shifted the λmax to 347 nm. At 8.0 M urea, the fluorescence intensity was decreased again with a shift of λmax to 352 nm indicating the complete unfolding of the protein. The present results indicate that three intermediates could be identified between the native dimer and unfolded monomer

fied for BaGSTM.

Applications

274

are 218, atoms 1765, and bonds 1813 [35, 36].

(third phase) [33].

during the unfolding of BaGSTM.

**Figure 4.** Fluorescence–emission spectra of BaGSTM (20 µg/ml) equilibrated in buffer A (20 mM potassium phosphate buffer, pH 7.0 containing 1 mM EDTA/1 mM dithiothreitol) at different GdmCl concentrations ranging from 0 to 4.0 M at room temperature. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm (a to e). Unfolding was expressed as the ratio of fluorescence at 329 nm to the fluorescence at 352 nm (f).

4 **Figure 4.** Fluorescence–emission spectra of BaGSTM (20 µg/ml) equilibrated in buffer A (20 mM 5 potassium phosphate buffer, pH 7.0 containing 1 mM EDTA/1 mM dithiothreitol) at 6 different GdmCl concentrations ranging from 0 to 4.0 M at room temperature. Excitation was done at

1

2

6

12 Book Title

Running Title

**a**

**c pH 2.2**

**Relative Fluorescence Intensity**

**Relative fluorescence intensity**

1

2 3

8

15 proteins.

many monomeric proteins.

**300 320 340 360 380 400**

**Wavelength (nm)**

**300 320 340 360 380 400**

**Wavelength (nm)**

**pH 1.2**

**pH 7.0**

termined using the same excitation wavelength (d).

**pH 2.6**

**pH 7.0, 6.2, 5.8 and 5.4 pH 5.0**

> **pH 1.6 pH 2.0**

13

277

**pH 7.0, 4.6 and 4.2**

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482

**300 320 340 360 380 400**

**Wavelength (nm)**

**d**

**2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0**

**pH**

**pH 3.4 pH 3.8**

**pH 3.0**

**b**

**Relative fluorescence intensity**

**Emissin maximium (nm)**

 **Figure 6.** pH-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein (20μg/ml) was equilibrated at room temperature in 0.02 M citrate-phosphate buffer at pH from 7.0 to 1.2 for 1 h before measurement. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm (a-c). Emission maxima were determined using the same excitation wavelength (d).

**Figure 6.** pH-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein (20μg/ml) was equi‐ librated at room temperature in 0.02 M citrate-phosphate buffer at pH from 7.0 to 1.2 for 1 h before measurement. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm (a-c). Emission maxima were de‐

However, between pH 5.0 and 3.5, an initial red shift, from 338 to 342 nm of the maximum fluorescence emission, indicates a partial exposure of one or more tryptophanyl residues to the solvent. From pH 3.5 to pH 2.0 a second fluorescence transition occurs, characterized by a blue shift of λmax to 337 nm. This indicates the formation of a new type of structure in which the environment of the tryptophanyl residues is more hydrophobic. It has been pro‐ posed that the molten globule represents a common intermediate of the acid denaturation of

GSTs are crystallized as dimers, but in solution class mu GST from rat its Asp97 mutant en‐ zymes undergo reversible association and dissociation, the extent of which depends on pro‐ tein concentration. Addition of 3 M potassium bromide to buffer solutions containing the wild-type rGSTM1-1 has generated monomers (GSTM1) [37]. A monomeric species of a hu‐ man GSTpi has been constructed by introducing 10 site specific mutations. This drastically

The nonsubstrate ligand 8-anilino-1-naphthalene sulfonate (ANS) is a negatively charged hydrophobic fluorescent molecule, largely used to check the presence of compact partially folded intermediates. In fact, its very low fluorescence quantum yield in polar environment is strongly increased in non polar solvents [39]. Therefore, the binding of this molecule to

changed enzyme was structurally stable, but retained no activity [38].

 However, between pH 5.0 and 3.5, an initial red shift, from 338 to 342 nm of the maximum fluorescence emission, indicates a partial exposure of one or more tryptophanyl residues to the solvent. From pH 3.5 to pH 2.0 a second fluorescence transition occurs, characterized by a blue shift of λmax to 337 nm. This indicates that at pH 2.0 there is the formation of a new type of structure in which the environment of the tryptophanyl residues is more hydrophobic. It has been proposed that the molten globule represents a common intermediate of the acid denaturation of many monomeric

7 In particular, the pH-dependent fluorescence transition of BaGSTM is clearly characterized by many 8 distinct steps. The behavior of the protein in an acidic environment was investigated and analyses of 9 fluorescence emission spectra of BaGSTM in solutions at different pH values were performed. The 10 position of the emission maximum of a protein's fluorescence spectrum, upon excitation at 280 nm, **Figure 5.** Urea-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein (20µg/ml) was equilibrated in buffer A in the presence of the indicated concentration of urea at room temperature. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm.

11 was highly sensitive to the environment around its tryptophanyl and tyrosyl residues. As shown in

3 **Figure 5.** Urea-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein 4 (20µg/ml) was equilibrated in buffer A in the presence of the indicated concentration of urea at room 5 temperature. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm.

12 figure. 6, the acid denaturation of BaGSTM, as followed by the intrinsic fluorescence changes, was 13 characterized by the presence of at least three transition states [33]. In particular, the pH-dependent fluorescence transition of BaGSTM is clearly characterized by many distinct steps. The behavior of the protein in an acidic environment was investigat‐ ed and analyses of fluorescence emission spectra of BaGSTM in solutions at different pH values were performed. The position of the emission maximum of a protein's fluorescence spectrum, upon excitation at 280 nm, was highly sensitive to the environment around its tryptophanyl and tyrosyl residues. As shown in figure 6, the acid denaturation of BaGSTM, as followed by the intrinsic fluorescence changes, was characterized by the presence of at least three transition states [33].

Running Title

1

2

12 Book Title

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**b**

**Relative fluorescence intensity**

**Relative fluorescence intensity**

3 **Figure 5.** Urea-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein 4 (20µg/ml) was equilibrated in buffer A in the presence of the indicated concentration of urea at room 5 temperature. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm.

 In particular, the pH-dependent fluorescence transition of BaGSTM is clearly characterized by many distinct steps. The behavior of the protein in an acidic environment was investigated and analyses of fluorescence emission spectra of BaGSTM in solutions at different pH values were performed. The position of the emission maximum of a protein's fluorescence spectrum, upon excitation at 280 nm, was highly sensitive to the environment around its tryptophanyl and tyrosyl residues. As shown in figure. 6, the acid denaturation of BaGSTM, as followed by the intrinsic fluorescence changes, was

**Figure 5.** Urea-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein (20µg/ml) was equilibrated in buffer A in the presence of the indicated concentration of urea at room temperature. Excitation was

In particular, the pH-dependent fluorescence transition of BaGSTM is clearly characterized by many distinct steps. The behavior of the protein in an acidic environment was investigat‐ ed and analyses of fluorescence emission spectra of BaGSTM in solutions at different pH values were performed. The position of the emission maximum of a protein's fluorescence spectrum, upon excitation at 280 nm, was highly sensitive to the environment around its tryptophanyl and tyrosyl residues. As shown in figure 6, the acid denaturation of BaGSTM, as followed by the intrinsic fluorescence changes, was characterized by the presence of at

**300 320 340 360 380 400**

**Wavelength (nm)**

**<sup>d</sup> 5.5, 6.0 & 6.5 M**

**8.0 M**

**None**

**7.0 M**

**300 320 340 360 380 400**

**Wavelength (nm)**

**2.0 & 2.25 M**

**1.75 M**

**None**

**2.75 & 3.0 M 2.5 M**

**5.0 M**

**300 320 340 360 380 400**

**Wavelength (nm)**

**4.25 M 4.5 M**

**300 320 340 360 380 400**

**Wavelength (nm)**

13 characterized by the presence of at least three transition states [33].

done at 280 nm and fluorescence was recorded from 300 to 400 nm.

least three transition states [33].

**3.25 M**

**None**

**4.0 M**

**None**

**0.5 & 0.75 M 1.0 M 1.2 M 1.5 M**

**3.5 & 3.75 M**

**a**

**c**

**Relative fluorescence Intensity**

**Relative fluorescence intensity**

1

Applications

276

2

6

3 4 **Figure 6.** pH-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein 5 (20μg/ml) was equilibrated at room temperature in 0.02 M citrate-phosphate buffer at pH from 7.0 to 6 1.2 for 1 h before measurement. Excitation was done at 280 nm and fluorescence was recorded from **Figure 6.** pH-induced equilibrium unfolding for BaGSTM monitored by fluorescence. The protein (20μg/ml) was equi‐ librated at room temperature in 0.02 M citrate-phosphate buffer at pH from 7.0 to 1.2 for 1 h before measurement. Excitation was done at 280 nm and fluorescence was recorded from 300 to 400 nm (a-c). Emission maxima were de‐ termined using the same excitation wavelength (d).

7 300 to 400 nm (a-c). Emission maxima were determined using the same excitation wavelength (d).

8 9 However, between pH 5.0 and 3.5, an initial red shift, from 338 to 342 nm of the maximum 10 fluorescence emission, indicates a partial exposure of one or more tryptophanyl residues to the 11 solvent. From pH 3.5 to pH 2.0 a second fluorescence transition occurs, characterized by a blue shift 12 of λmax to 337 nm. This indicates that at pH 2.0 there is the formation of a new type of structure in 13 which the environment of the tryptophanyl residues is more hydrophobic. It has been proposed that 14 the molten globule represents a common intermediate of the acid denaturation of many monomeric 15 proteins. However, between pH 5.0 and 3.5, an initial red shift, from 338 to 342 nm of the maximum fluorescence emission, indicates a partial exposure of one or more tryptophanyl residues to the solvent. From pH 3.5 to pH 2.0 a second fluorescence transition occurs, characterized by a blue shift of λmax to 337 nm. This indicates the formation of a new type of structure in which the environment of the tryptophanyl residues is more hydrophobic. It has been pro‐ posed that the molten globule represents a common intermediate of the acid denaturation of many monomeric proteins.

GSTs are crystallized as dimers, but in solution class mu GST from rat its Asp97 mutant en‐ zymes undergo reversible association and dissociation, the extent of which depends on pro‐ tein concentration. Addition of 3 M potassium bromide to buffer solutions containing the wild-type rGSTM1-1 has generated monomers (GSTM1) [37]. A monomeric species of a hu‐ man GSTpi has been constructed by introducing 10 site specific mutations. This drastically changed enzyme was structurally stable, but retained no activity [38].

The nonsubstrate ligand 8-anilino-1-naphthalene sulfonate (ANS) is a negatively charged hydrophobic fluorescent molecule, largely used to check the presence of compact partially folded intermediates. In fact, its very low fluorescence quantum yield in polar environment is strongly increased in non polar solvents [39]. Therefore, the binding of this molecule to

partially folded proteins, containing clusters of hydrophobic side chains accessible to sol‐ vent, is often observed in the presence of molten globules [23].

The unfolding results of the *R. (Boophilus) annulatus* (BaGSTM) demonstrate that the probes used (intrinsic fluorescence, excitation of tryptophan and tyrosine and ANS binding fluores‐ cence) are differentially sensitive to various conformational states of BaGSTM. The presence of multiple nonsuperimposable transitions for this enzyme indicates that the two states un‐ folding mechanism is not applicable, and it is highly suggestion of the existence of at least two well-populated stable intermediates. Similar data were reported demonstrating the exis‐ tence of stable intermediates at the unfolding of GSTs [42, 45-48]. However, only one transi‐ tion intermediate was detected using ANS binding fluorescence for BaGSTM in presence of different concentrations of GdmCl or urea. This result is similar to that observed for sigma

Glutathione *S*-Transferase Genes from Ticks http://dx.doi.org/10.5772/52482 279

Tick control is considered crucial all over the world to minimize the major drawbacks of tick infestations. The control strategies were adopted to use the acaricides which become less ef‐ ficient. Seixas et al. [49] has stated the alternative approaches used in tick control and are classified into four groups: (i) biological control by tick pathogens or predators [50]; (ii) habi‐ tat alterations by planting tick-killing or repelling vegetation [51]; (iii) immunological con‐ trol [52]; and (iv) development of tick-resistant breeds. They stated that although these methods have been proved to be theoretically valuable, most of them have been forsaken, since they did not afford acceptable cost/benefit ratios under field conditions, except for vac‐

Many types of chemical tick control were started since 1893 including the application of ar‐ senic acaricides, gamma BHC (organochlorine acaricide) and DDT. However, resistance to DDT was reported within 5 years of field application [53]. The toxicity plus the environmen‐ tal awareness and other factors led to the removal of the previously mentioned acaricides

In early 1960's, Diazinon and other organophosphate acaricides were applied and in 1970's, Triatix was the first amidine to be registered for tick control. In the 1980's, pyrethroids (Flu‐ methrin and Permethrin) were also registered for tick control. The following table 2 shows

Along with the application of these chemicals with acaricidal properties as the predominant method of tick control throughout the world, resistance to the majority of the groups of chemicals had been evolved. Generally, the resistance may arise through several mecha‐ nisms including target sites, metabolic mechanism or reduced penetration. The target site re‐ sistance occurs when an allele of the gene coding for the target molecule is attacked by the acaricide. The penetration resistance occurs when an acaricide fails to penetrate the target individual. This type of resistance has not been reported in ticks. The third type of resistance mechanisms is the metabolic pathway which occurs through changes in the abilities of acari‐ cide detoxification by an organism [17]. Three enzyme families including cytochrome P450s

class GST in the presence of urea [46].

**4. GST and immune system**

from the tick control market.

the different acaricides used in tick control.

cines.

Unbound ANS emission spectra showed a maximum at 530 nm that was blue shifted upon binding of the dye to the protein. Binding of ANS to BaGSTM as a function of GdmCl concen‐ tration showed one peak centered at 1.5 M and one peak as a function of urea concentrations centred at 3.5 M (Figures 7 a and b). ANS binding fluorescence of BaGSTM as a function of pH did not show any transition peak. However, the fluorescence intensity was increased as the pH decreased. The fluorescence intensity about 2000 fold higher at pH 2.0 compared with that at neutral pH (Figure 7 c). At the neutral pH, the fluorescence of ANS in the presence of BaGSTM is perfectly super imposable to that of ANS alone. At less than pH 3.8, ANS binds to BaGSTM showed a blue shift displacement with an enhancement of fluorescence intensity. Binding of the dye occurs at the dimer interface and unfolded GST does not bind ANS. This makes ANS an excellent probe to monitor changes at the packing of hydrophobic cores in protein which un‐ dergoes structural changes and has been broadly used to study the presence of monomeric in‐ termediates at the urea/GdmCl unfolding of several GSTs [38, 40-42]. ANS was also used to detect the presence of folding intermediates with hydrophobic patches such as the molten globule in penicillin G acylase [43] and apomyoglobin [44]. Running Title 15

1

2

7

3 **Figure 7.** Variation of ANS binding fluorescence at 480 nm as a function of GdmCl concentrations 4 (a) or urea (b) or as a function of pH (c). The proteins (20 μg/ml) were equilibrated in buffer A at 5 room temperature in the presence of the indicated concentration of denaturant or pH. 100 μM ANS 6 was added to the solution. Excitation was done at 380 nm and emission at 480 nm. **Figure 7.** Variation of ANS binding fluorescence at 480 nm as a function of GdmCl concentrations (a) or urea (b) or as a function of pH (c). The proteins (20 μg/ml) were equilibrated in buffer A at room temperature in the presence of the indicated concentration of denaturant or pH. 100 μM ANS was added to the solution. Excitation was done at 380 nm and emission at 480 nm.

 The unfolding results of the *R.* (Boophilus) *annulatus* (BaGSTM) demonstrate that the probes used (intrinsic fluorescence, excitation of tryptophan and tyrosine and ANS binding fluorescence) are differentially sensitive to various conformational states of BaGSTM. The presence of multiple nonsuperimposable transitions for this enzyme indicates that the two states unfolding mechanism is not applicable, and it is highly suggestion of the existence of at least two well-populated stable intermediates. Similar datra were reported demonstrating the existence of stable intermediates at the unfolding of GSTs [42, 45-48]. However, only one transition intermediate was detected using ANS binding fluorescence for BaGSTM in presence of different concentrations of GdmCl or urea. This

16 result is similar to that observed for sigma class GST in the presence of urea [46].

The unfolding results of the *R. (Boophilus) annulatus* (BaGSTM) demonstrate that the probes used (intrinsic fluorescence, excitation of tryptophan and tyrosine and ANS binding fluores‐ cence) are differentially sensitive to various conformational states of BaGSTM. The presence of multiple nonsuperimposable transitions for this enzyme indicates that the two states un‐ folding mechanism is not applicable, and it is highly suggestion of the existence of at least two well-populated stable intermediates. Similar data were reported demonstrating the exis‐ tence of stable intermediates at the unfolding of GSTs [42, 45-48]. However, only one transi‐ tion intermediate was detected using ANS binding fluorescence for BaGSTM in presence of different concentrations of GdmCl or urea. This result is similar to that observed for sigma class GST in the presence of urea [46].
