High-Temperature unfolding of a trp-Cage mini-protein: a molecular dynamics simulation study
© Seshasayee; licensee BioMed Central Ltd. 2005
Received: 09 October 2004
Accepted: 11 March 2005
Published: 11 March 2005
Trp cage is a recently-constructed fast-folding miniprotein. It consists of a short helix, a 3,10 helix and a C-terminal poly-proline that packs against a Trp in the alpha helix. It is known to fold within 4 ns.
High-temperature unfolding molecular dynamics simulations of the Trp cage miniprotein have been carried out in explicit water using the OPLS-AA force-field incorporated in the program GROMACS. The radius of gyration (Rg) and Root Mean Square Deviation (RMSD) have been used as order parameters to follow the unfolding process. Distributions of Rg were used to identify ensembles.
Three ensembles could be identified. While the native-state ensemble shows an Rg distribution that is slightly skewed, the second ensemble, which is presumably the Transition State Ensemble (TSE), shows an excellent fit. The denatured ensemble shows large fluctuations, but a Gaussian curve could be fitted. This means that the unfolding process is two-state. Representative structures from each of these ensembles are presented here.
Understanding the mechanisms behind protein folding, which is one of the most fundamental biochemical processes, is proving to be a challenging task for biochemists and biophysicists. Recent developments in instrumentation and methodology have enabled us to take major steps forward in comprehending the dynamics of proteins and peptides at the molecular level. Protein engineering methods such as Phi-value analysis  and various spectroscopic techniques such as NMR have made the task more practicable.
Proteins are composed of two major secondary structural elements, helices and sheets, which, along with loops, pack together to form super-secondary and tertiary structures. Trp cage is a novel, and a highly stable, mini-protein fold. A 20-residue Trp-cage miniprotein has been designed . It has the sequence NLYIQWLKDGGPSSGRPPPS. While residues 1–9 form an alpha helix, residues 10–15 form a 3,10 helix. W6 is caged by the C-terminal poly-proline stretch. D9 and R16 are involved in a stabilizing salt-bridge interaction.
Molecular dynamics simulations, which make use of classical Newton mechanics to generate trajectories, are playing an ever-expanding role in biochemistry and biophysics due to substantial increases in computational power and concomitant improvements in force fields. In particular, the contribution of such studies to protein folding is immense . As pointed out by Fersht and Dagget, molecular dynamics simulations are capable of unraveling whole protein folding / unfolding pathways . Indeed, simulation techniques have been widely used for studying helices and sheets. Today, folding simulations of more-than-model peptides are being carried out on high-power computers.
Despite being a new mini-protein construct, the Trp cage motif has attracted considerable computational analysis. Folding simulations of this protein in explicit water have been carried out using what is known as the Replica Exchange Method. A two-state folding mechanism has been proposed and free energy surfaces have been determined . Moreover, a few folding simulations of have been carried out using implicit solvation models [4–6]. In this article, the results of a high-temperature unfolding simulation of the Trp-cage mini-construct are presented. Three separate structural clusters are identified: the close-to-native-state cluster, the intermediate cluster and the denatured ensemble. These clusters, considered in terms of their radii of gyration, are shown to be Gaussian ensembles. Structural features representing each of these ensembles are also illustrated.
Results and Discussion
Molecular dynamics simulations of the Trp-cage mini-protein construct (PDB ID: 1L2Y) were carried out using the OPLS-AA force-field incorporated in the freely available program, GROMACS. The simulations were carried out at 498 K, at which temperature the unfolding process is favored. This temperature provides a good description of the unfolding process, at least in respect of CI2 and the homeodomain of engrailed . It is also much higher than the melting temperature determined by experiment (315 K) or through replica-exchange simulations (400 K) .
After 3200 ps, a further jump in RMSD and Rg is observed leading to a state where these values fluctuate markedly. This highly disordered state, showing a measure of heterogeneity, is the denatured ensemble, in which the salt-bridge interaction that characterized the intermediate state is also lost. There is a significant jump in the distance between the Asp9 and Arg 16 sidechains after this time. As a result, there are no native contacts in this state. This is represented by structures in figures 3e and 3f.
Rg range and time corresponding to each state seen in the simulation
Rg range (nm)
0.7 – 0.8
0.72 – 1
0.8 – 1.4
0.7 – 1.4
High-temperature unfolding molecular dynamics simulations of a Trp cage miniprotein construct have been carried out. This has shown that the process is two-stage, akin to the folding process results . The three ensembles, including the TSE, are shown to be Gaussian with respect to their Rg values.
The starting structures for the simulations were obtained from PDB 1L2Y . The first three models were used to carry out the 5 ns simulations and similar results were obtained with each. Results presented here correspond to model 1. All simulations were carried out using GROMACS 3.2 [8, 9], running on a single Fedora Linux system. The OPLS-AA force field was used. The peptide was solvated in a box containing approx. 500 water molecules . Periodic boundary conditions were employed to eliminate surface effects. Energy minimization with a tolerance of 2000 kJ/mol/nm was carried out using the Steepest Descent method. All bonds were constrained using LINCS . The system was loosely coupled to a temperature bath (at 498 K or 293 K) using Berendsen's method . Berendsen's pressure coupling was used. Long-range electrostatics was handled using the PME method . All potential cut-offs were set at 1 nm. The final MD simulations were carried out with a time-step of 2 fs and without any position restraints. All analyses were conducted using programs built within GROMACS. The RMSD values were obtained from a least square fit of the respective non-hydrogen atoms (main-chain and side-chain). The radius of gyration was also calculated for the whole protein minus hydrogens as an indicator of the compactness of the overall structure. The compiled DSSP , which was downloaded separately and run from GROMACS, was used to calculate secondary structure formation.
I would like to thank Prof. P. Gautam of Centre for Biotechnology, Anna University for being a constant source of inspiration and encouragement. I also thank Mr. Mahesh Viswanathan for helping me with drawing the graphs. I also thank the anonymous reviewers for their comments.
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