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Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation

[ Vol. 24 , Issue. 11 ]

Author(s):

Bhaswati Sengupta, Apala Chaudhury, Nilimesh Das and Pratik Sen*   Pages 1073 - 1081 ( 9 )

Abstract:


Introduction: Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest.

Objective: The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant.

Methods: Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment.

Results: It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation.

Conclusion: It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state.

Keywords:

Papain, chemical denaturation, folding intermediate, fluorescence correlation spectroscopy, conformational dynamics, protease enzyme.

Affiliation:

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur -208 016, UP, Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur -208 016, UP, Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur -208 016, UP, Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur -208 016, UP

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