Identification of putative unfolding intermediates of the mutant His‐107‐tyr of human carbonic anhydrase II in a multidimensional property space

Abstract

In this article, we develop an extensive search procedure of the multi-dimensional folding energy landscape of a protein. Our aim is to identify different classes of structures that have different aggregation propensities and catalytic activity. Following earlier studies by Daggett et al. [Jong, D. D.; Riley, R.: Alonso, D.O.: Dagett, V. J. Mol. Biol. 2002, 319, 229], a series of high temperature all-atom classical molecular simulation studies has been carried out to derive a multi-dimensional property space. Dynamical changes in these properties are then monitored by projecting them along a one-dimensional reaction coordinate, dmean . We have focused on the application of this method to partition a wide array of conformations of wild type human carbonic anhydrase II (HCA II) and its unstable mutant His-107-Tyr along dmean by sampling a 35-dimensional property space. The resultant partitioning not only reveals the distribution of conformations corresponding to stable structures of HCA II and its mutant, but also allows the monitoring of several partially unfolded and less stable conformations of the mutant. We have investigated the population of these conformations at different stages of unfolding and collected separate sets of structures that are widely separated in the property space. The dynamical diversity of these sets are examined in terms of the loading of their respective first principal component. The partially unfolded structures thus collected are qualitatively mapped on to the experimentally postulated light molten globule (MGL) and molten globule (MG) intermediates with distinct aggregation propensities and catalytic activities. Proteins 2016; 84:726-743. © 2016 Wiley Periodicals, Inc.

Unfolding intermediates of the mutant His-107-Tyr of human carbonic anhydrase II

“…Detailed methodology of generating the high temperature unfolding trajectories for the mutant His-107-Tyr has been discussed earlier 12 and is briefly outlined here.…”

“…(1) Trajectory wt: wild type enzyme at 300 K, (2) Trajectory mut: the mutant His-107-Tyr at 277 K where it is expected to be most stable, and (3) Trajectories mut-ht: the mutant His-107-Tyr simulated at 500 K under four different conditions of heating to 500 K. 12 Each set of trajectory has been generated from an input model structure, solvated, energy minimized and heated to a desired temperature in a large cubic box with around 15,000 TIP3P 15 water molecules with periodic boundary conditions using the solvation utilities of NAMD. 13 In the next step, 1 ns of equilibration and 50 ns of production runs are carried out under isothermal-isobaric conditions using CHARMM22 16 force field parameters.…”

“…A complete list is available in Ref. 12 The extent of unfolding of a given structure is conventionally monitored in terms of structural parameters such as RMSD, R g and solvent accessible surface area (SASA). We have used the change in SASA (compared to wt or mut) as one of the indicators of increasing aggregation propensity displayed by a given structure.…”

“…Therefore, a computational methodology has been designed and described in Ref. 12 to address this issue specifically. Different steps adopted in this method are schematically shown in Figure 2 and their salient features are summarized below.…”

“…Following the above method, four distinct sets of structures, A, B, C1 and C2 are detected as putative unfolding intermediates of the mutant His-107-Tyr of HCA II. 12 The structures belonging to set A are least unfolded and least prone to aggregation. They are found to have a native-like active site to some extent.…”