The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. Detailed knowledge of the thermal stability of proteins and the phenomenon of protein folding are essential not only to understand protein structure and function but also to design thermostable proteins for industrial applications [
Barnase (E.C.3.1.2.7) is an extracellular 110 residue ribonuclease from
The small size of the protein, the absence of metal cofactors, and the absence of disulfide bonds make Barnase an ideal test case for studying the contribution of salt bridge to thermostability by computer simulation. In this paper we have performed molecular dynamics simulation of Barnase at five different temperatures, namely, 300 K, 400 K, 500 K, 550 K, and 600 K. By calculating the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values for backbone and C
Molecular dynamics simulations [
Empirical energy functions can only be used to approach the force fields for large biological systems composed of many atoms. Here the CHARMM (Chemistry at HARvard Macromolecular Mechanics) force field is selected, which has been developed and continuously revised to better match new experimental data for over 25 years.
The potential function is a sum of some interaction energies. The value of the potential is determined by summing the bonded terms
The nonbonded terms calculation in the potential function is the most time-consuming part of the MD simulation. Generally, the interactions between every pair of atoms should be calculated definitely, meaning that, for an N-atom system,
The Langevin dynamics [
From classic theories on Brownian motion, it can be seen that, although molecular collisions are random, the ensemble of these collisions produces a systematic effect. That is to say, the molecular random motions exist at thermal equilibrium as a fluctuation. Hence one can see that the frictional force is a correlate of the random force by the fluctuation/dissipation theorem [
The Dirac-
The random force is chosen independently for each step. And the covariance matrix is diagonal as hydrodynamic interactions between particles have been discounted.
A physical value of
The damping coefficient
For constant pressure and temperature simulations in which Langevin dynamics are used to control temperature, the pressure can be controlled in NAMD with a modified Nose-Hoover method. This method entitled Nose-Hoover [
The internal virial vir is proportional to the inner product of the each atom’s position vector
The conserved quantity under these augmented equations of motion is
By this means, the magnitude of the system fluctuates under specified thermostat and barostats, and the system is driven to steady state at which the average internal pressure
All simulations were performed on a PC with a Pentium 4 2.8 GHz dual core processor running Windows operating system and using the molecular dynamics program NAMD [ A Protein Data Bank (pdb) file which stores atomic coordinates and/or velocities for the system is needed. The coordinates for starting configurations Barnase was obtained from the Protein Data Bank (PDB entry codes 1RNB [ A Protein Structure File (psf) which contains all of the molecule-specific information needed to apply a particular force field to a molecular system is needed. Coordinates of the atoms that were missing in the crystallographic structure were reconstructed using the PSFGEN structure building utility, a module of NAMD. The protein needs to be solvated and put inside water, to more closely resemble the cellular environment. The protein was solvated in a cubic box consisting of TIP3 water molecules [ We need a force field parameter file. A force field is a mathematical expression of the potential which atoms experience in the system. A CHARMM forcefield parameter file contains all of the numerical constants needed to evaluate forces and energies, given a PSF structure file and atomic coordinates. The CHARMM parameters are available for download from the website: Create the simulation script, in which we specified all the options that NAMD should adopt in running a simulation. NAMD parses its configuration file using the Tcl scripting language. First we specify the files that contain the molecular structure and initial conditions. Setting the Tcl variable temperature makes it easy to change the target temperature for many options. The outputName prefix will be used to create all of the trajectory, output, and restart files generated by NAMD run. Next is the parameter file itself and the options that control the nonbonded potential functions. These are mostly specified by the CHARMM force field. In force-field parameters, the cutoff distance was specified to 12 Å. Electrostatic interactions were calculated using the Particle Mesh Ewald (PME) summation scheme. Turn on switching for the van der Waals interactions, which were calculated with a switching function from 10 Å to 12 Å. SHAKE method [ The system was subjected to energy minimization for 1000 steps by steepest descents and subsequently equilibrated for 500 ps, and then the equilibrated system was subjected to molecular dynamics simulations for 2 ns each at five different temperatures, namely, 300 K, 400 K, 500 K, 550 K, and 600 K. The coordinates were saved at every 500 time steps.
MD simulations generate an ensemble of conformations and thus include valuable information of the protein dynamics. In the following we present a detailed analysis of the four molecular dynamics trajectories, in water, generated for the protein of Barnase. The RMSD of the backbone atoms of the protein from the starting structure over the course of simulation may be used as a measure of the conformational stability of a protein during the simulation. The plots of RMSD of the protein versus time at different temperatures are shown in Figure
Time evolution. (a) Backbone RMSD of Barnase at different temperatures. (b) RMSFs as a function of residue number of Barnase at different temperatures. The color-coding scheme is as follows: 300 K (red), 400 K (blue), 500 K (magenta), and 600 K (cyan).
Following molecular dynamics simulations at multiple temperatures, it was of interest to determine which general regions of the Barnase polypeptide chain exhibit hypersensitivity to thermal stress. The results are shown in Figure
At high temperature simulations, dramatically increased RMSD values were observed for the loop regions and both terminal regions in Barnase, which indicate these regions extensive local conformational changes upon thermal unfolding. A close analysis of the time evolution of the secondary structure (Figure
Time evolution of secondary structure in unfolding trajectory at different temperatures for Barnase. (a) 300 K. (b) 400 K. (c) 500 K. (d) 550 K. The different secondary elements are presented in a color code format indicated at the bottom of the figure.
Figure
At 500 K, protein structure fluctuation is significantly more pronounced. In this time period the dominant structural change was the expansion of the protein in response to the temperature increase, and the packing density in the three hydrophobic cores decreased. During the simulation, the edge residues, particularly those at or near the N-terminal part of
Snapshots from the thermal unfolding simulations of Barnase. The structures are made with the VMD program,
When the temperature is increased to 550 K, the structure of the protein shows a continuous and progressive unfolding. The N terminus begins to unfold during the first 250 ps. This is followed by partial denaturation of
Hydrogen bond is one of the factors influencing the thermal stability of protein. In the hydrogen bond calculations a distance cutoff of 3.0 Å and an angle cutoff of 20° were applied. The average numbers of hydrogen bonds are 29, 24, 17, and 18 for the 300 K, 400 K, 500 K, and 600 K simulations, respectively. Thus, as the simulation temperature is increased, there is a concomitant decrease in number of intact hydrogen bonds. This is reasonable as the structures become more distorted as the simulation temperature is raised. It is also evident from the plot (Figure
Time evolution of hydrogen bonds of the protein at four different temperatures. (a) 300 K. (b) 400 K. (c) 500 K. (d) 600 K.
To further probe the stability behavior of Barnase under thermal stress, we analyzed another important intramolecular contact, namely, salt bridge. Charged residues in globular proteins frequently form salt bridges. The electrostatic contribution of salt bridge has been suggested to be important for protein stability. Furthermore, the statistical analysis of salt bridges from mesophilic and thermophilic organisms has shown a higher frequency of complex salt bridges in thermophilic proteins, suggesting that they have a special role in thermostabilization.
In the structure of Barnase, nine salt bridges, Asp8-Arg110, Asp12-Arg110, Asp54-Lys27, Glu73-Lys27, Asp75-Arg83, Asp75-Arg87, Asp93-Arg69, Asp93-Lys66, and Glu60-Lys62, can be identified with the help of VMD. Interestingly, among these salt bridges, there are four salt bridge networks. Networks of ionic interactions occur when more than two ionic residues interact, and an increased occurrence has been suggested to be essential in explaining the enhanced thermal stability of protein [
Oxygen-nitrogen distance of salt bridges plots. Time evolution of distance between selected atoms during simulation. (a) Distance between
The salt bridge network of Asp8-Arg110-Asp12 is located in the main hydrophobic core1, which is formed by the packing of
The rupture of the double salt bridge initiates the separation of the
In the 550 K simulation, ruptures and restorations of the salt bridge Asp8-Arg110 were observed along the first half of unfolding simulations, and the Asp8 and Arg110 side chains begin to recover during the 1110 to 1890 ps period. The separation of the Asp12 and Arg110 side chains begins at initial stage of simulation, and the side chains of two residues came within a relatively short distance during the 381 to 1005 ps and 1099 to 1500 ps period of the simulation, but they fell apart eventually. The denaturation of the N-terminal part of
The salt bridge network of Asp54-Lys27-Glu73 was found to be stable at 300 K and 400 K simulations (Figure
The double salt bridges between Arg83, Arg87, and Asp75 were found to be very stable, up to 500 K simulation (Figure
Another salt bridge network Lys66-Asp93-Arg69 is also close to the core3. In the network, the two residues Lys66 and Arg69 are all located in loop3, and the residue Asp93 is located in the
Finally, there is salt bridge Glu60-Lys62 located in the outer domain of hydrophobic core3, and the two residues are all located in loop3. Loop residues showed increased mobility relative to sheet or helical residues (Figure
On the basis of these results, we concluded that surface salt bridge does stabilize the native state of the protein at elevated temperatures, and the complex salt bridge contribution to the overall protein stability is more than the individual pairs. Several experiments also confirm that hyperthermophilic proteins generally possess not only an increased number of surface salt bridges, but also an increased number of salt bridge networks [
We have here investigated the electrostatic stability of noncovalent interactions in the context of temperature adaptation of Barnase by using MD simulations. The results show that hydrogen bond is very sensitive to heat, while salt bridge is comparatively stable. Nine salt bridges have been identified (Asp8-Arg110, Asp12-Arg110, Asp54-Lys27, Glu73-Lys27, Asp75-Arg83, Asp75-Arg87, Asp93-Arg69, Asp93-Lys66, and Glu60-Lys62) as critical salt bridges. These salt bridges are of fundamental importance in maintaining the structural integrity of the protein structure. Among these nine pairs, two salt bridge networks (Arg83-Asp75-Arg87 and Lys66-Asp93-Arg69) have been found to be extremely stable throughout the simulation up to 500 K. Two salt bridge networks located in core3 outer domain add more stability towards thermostable core3 region. The strength and the number of salt bridges present in a protein and whether they are involved in networks or not are important for the overall structural stability. Their presence can have a large impact on the structural integrity modulating molecular plasticity. The present study attempts to gain a deeper understanding of the noncovalent intramolecular interaction factors conferring thermostability of Barnase. It can offer a general picture of the first steps of unfolding and may help to design biotechnologically improved thermostable proteins.
This work was supported in part by the Fundamental Research Funds for the Central Universities under Grant no. JUSRP111A46 and the National Natural Science Foundation of China under Grant no. 61170119. Ming Li thanks the supports in part by the National Natural Science Foundation of China under the Project Grant nos. 61272402, 61070214, and 60873264.