Increasing Temperature Accelerates Protein Unfolding Without Changing the Pathway of Unfolding

Abstract

We have traditionally relied on extremely elevated temperatures (498 K, 225 8C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 ms. At 298 K, the protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

“…While there is evidence that MD simulations at elevated temperature adequately represent the unfolding events, [76][77][78] it is clearly desirable to conduct the simulations under milder conditions. Furthermore, introducing solvent and electrostatic interactions is key to more realistic modeling of the PRE effect (as discussed later, however, our vacuum simulations reproduced certain essential features that are normally associated with protein solvation).…”

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

“…The unfolding rate increases much more dramatically with temperature than the folding rate (Fersht 1999). In molecular simulations at elevated temperatures protein unfolding can occur within a few ns (Daggett 2002;Day et al 2002). Relative to the simulation time, the folding reaction is very slow at any reasonable temperature.…”

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

“…We compare the physical parameter (RMSD, SASA and Rg) of wild type and mutants to represent the thermostability of both molecules (Gundampati et al, 2013). Significant increase in Solvent Accessible Surface Area (SASA) will happen when a protein molecule starts to lose its stabilizing interactions (intermolecular hydrogen bond, electrostatic and van der Waals interaction) and hydrophobic core of protein molecule starts to collapse; thus the protein will be more readily accessible to the solvent molecule (Day et al, 2002;Yan et al, 2010). According to this explanation, more linear SASA value shows more stability in the protein structure along simulation time.…”

“…According to this explanation, more linear SASA value shows more stability in the protein structure along simulation time. Radius of gyration (Rg) can also be used to relate protein stability in terms of unfolding process (Li and Daggett, 1994;Day et al, 2002;Zhao et al, 2009;Paschek et al, 2011).…”

“…Computer aided design has become a common practice now in protein engineering, especially as an initial step to design a mutant enzyme and to simulate certain properties of the mutant (Yamaguchi et al, 1996;Santarossa et al, 2005;Sandström et al, 2009;Han et al, 2009;Kim et al, 2010). Molecular dynamic simulation helps researcher to study the physical changes in protein in their response to temperature changes (Li and Daggett, 1994;Day et al, 2002;Liu and Wang, 2003;Purmonen et al, 2007;Paschek et al, 2011). Analysis of flexible area in protein during temperature changes can be performed by molecular dynamic simulation.…”