TY - JOUR
T1 - GdnHCl-induced unfolding intermediate in the mitochondrial carbonic anhydrase VA
AU - Idrees, Danish
AU - Prakash, Amresh
AU - Haque, Md Anzarul
AU - Islam, Asimul
AU - Hassan, Md Imtaiyaz
AU - Ahmad, Faizan
N1 - Funding Information:
This work is supported by the grants from Council of Scientific and Industrial Research (Grant No. 37(1665)/15/EMR-II ). DI, AP and MAH are thankful to University Grants Commission (UGC), New Delhi, India, for awarding their fellowship. Harvard University-plasmid facility is acknowledged providing the CAVA gene. We thank Department of Science and Technology, India for FIST support (SR/FST/LSI-541/2012).
Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/10/1
Y1 - 2016/10/1
N2 - Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25 °C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25 M to 3.40 M of the denaturant. However, CAVA gets completely denatured at 4.0 M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40 ns were carried out and a well-defined correlation was established for both in vitro and in silico studies.
AB - Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25 °C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25 M to 3.40 M of the denaturant. However, CAVA gets completely denatured at 4.0 M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40 ns were carried out and a well-defined correlation was established for both in vitro and in silico studies.
KW - 1-Anilinonaphthalene-8-sulfonic acid
KW - Carbonic anhydrase VA
KW - GdnHCl-induced denaturation
KW - Molecular dynamics simulation
KW - Protein folding
UR - http://www.scopus.com/inward/record.url?scp=84978301490&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84978301490&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2016.06.080
DO - 10.1016/j.ijbiomac.2016.06.080
M3 - Article
C2 - 27365118
AN - SCOPUS:84978301490
SN - 0141-8130
VL - 91
SP - 1151
EP - 1160
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -