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Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin.

Ahmad E, Rabbani G, Zaidi N, Ahmad B, Khan RH - PLoS ONE (2012)

Bottom Line: These findings were compared to HSA-hydrolase activity.We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators.We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.

ABSTRACT
Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

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Stern-Volmer plot.Acrylamide quenching of native, denatured and pollutant complexed HSA (2 µM) at 37°C.
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pone-0038372-g004: Stern-Volmer plot.Acrylamide quenching of native, denatured and pollutant complexed HSA (2 µM) at 37°C.

Mentions: As HSA possesses a single Trp214 located on one side of the binding pocket in the second α-helix of subdomain IIA, it is feasible to study the accessibility to this moiety by a collisional fluorescence quencher, acrylamide, which can indicate the structural transitions induced by pollutants binding to albumin. Upon pollutant interaction with albumin, the extent of burial or exposure of Trp214 residue to the solvent was accordingly determined by monitoring the fluorescence emission intensity on increasing concentration of acrylamide as shown in the Figure 4. There were not any structural changes in albumin by acrylamide itself as the position of the emission peak was unaffected. The Stern-Volmer quenching constants of native and denatured albumin were taken as two extreme references (Table 2). From the obtained Ksv values, the values of kQ were calculated by taking the average integral lifetime of HSA Trp214 fluorescence as 4.3 ns [17]. The values of kQ were lower than the limiting diffusion constant Kdiff of the biomolecules (2×1010 M−1s−1) suggesting a dynamic quenching mechanism by the specific interaction of acrylamide with HSA. The straight line of the Stern-Volmer plot also implies that the quenching takes place on a simple collisional or dynamic basis in accordance with previous reports [18]. It was found that native HSA possess a smaller Stern-Volmer quenching constant (4.17 M−1) as well as lower accessibility of acrylamide to Trp214 than the denatured protein molecule (6.30 M−1) which indicates that Trp214 is located within the protein matrix. The addition of increasing concentrations of pollutants led to lower level of quenching even less than the native protein (Table 2). 1∶50 ([Protein]:[Ligand]) of 1N and 2N showed the maximum effect. Such changes are characteristic of a decrease in the polarity of Trp214environment. These results suggest that the structure of the protein matrix nearby Trp214 has become denser or more compact. The minimum amount of quenching was observed in the case of 8H in comparison to 1N and 2N. This was clear from the Ksv values, given in Table 2, which indicate that although these ligands did not result in decreased accessibility of Trp214 to the aqueous solvent, they did not denature the protein molecule.


Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin.

Ahmad E, Rabbani G, Zaidi N, Ahmad B, Khan RH - PLoS ONE (2012)

Stern-Volmer plot.Acrylamide quenching of native, denatured and pollutant complexed HSA (2 µM) at 37°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369883&req=5

pone-0038372-g004: Stern-Volmer plot.Acrylamide quenching of native, denatured and pollutant complexed HSA (2 µM) at 37°C.
Mentions: As HSA possesses a single Trp214 located on one side of the binding pocket in the second α-helix of subdomain IIA, it is feasible to study the accessibility to this moiety by a collisional fluorescence quencher, acrylamide, which can indicate the structural transitions induced by pollutants binding to albumin. Upon pollutant interaction with albumin, the extent of burial or exposure of Trp214 residue to the solvent was accordingly determined by monitoring the fluorescence emission intensity on increasing concentration of acrylamide as shown in the Figure 4. There were not any structural changes in albumin by acrylamide itself as the position of the emission peak was unaffected. The Stern-Volmer quenching constants of native and denatured albumin were taken as two extreme references (Table 2). From the obtained Ksv values, the values of kQ were calculated by taking the average integral lifetime of HSA Trp214 fluorescence as 4.3 ns [17]. The values of kQ were lower than the limiting diffusion constant Kdiff of the biomolecules (2×1010 M−1s−1) suggesting a dynamic quenching mechanism by the specific interaction of acrylamide with HSA. The straight line of the Stern-Volmer plot also implies that the quenching takes place on a simple collisional or dynamic basis in accordance with previous reports [18]. It was found that native HSA possess a smaller Stern-Volmer quenching constant (4.17 M−1) as well as lower accessibility of acrylamide to Trp214 than the denatured protein molecule (6.30 M−1) which indicates that Trp214 is located within the protein matrix. The addition of increasing concentrations of pollutants led to lower level of quenching even less than the native protein (Table 2). 1∶50 ([Protein]:[Ligand]) of 1N and 2N showed the maximum effect. Such changes are characteristic of a decrease in the polarity of Trp214environment. These results suggest that the structure of the protein matrix nearby Trp214 has become denser or more compact. The minimum amount of quenching was observed in the case of 8H in comparison to 1N and 2N. This was clear from the Ksv values, given in Table 2, which indicate that although these ligands did not result in decreased accessibility of Trp214 to the aqueous solvent, they did not denature the protein molecule.

Bottom Line: These findings were compared to HSA-hydrolase activity.We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators.We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.

ABSTRACT
Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

Show MeSH