Abstract

Thermal, chemical and force induced unfolding are three primary experimental approaches that have been successfully used to study protein folding and associated pathways. Recent experimental studies have demonstrated the importance of the use of combination of these techniques to study various aspects of protein folding. We have addressed this issue using two different model systems. The effect of temperature on the mechanical unfolding of titin I27 immunoglobulin domain has been investigated using steered molecular dynamics (MD) simulations performed both at constant velocity and constant force. The maximum force of unfolding decreases with respect to the increase in the temperature, but the extension corresponding to this force remains similar. However, we observed that the maximum probable unfolding force steadily decreases with an increase in temperature (see below). Several computations done on each of the temperatures consistently reproduce these effects. The results will be discussed in light of the available experimental data, and molecular level details on the conformational changes that effect such temperature dependence will be presented. In another study, we have done rigorous free energy calculations on the unfolding of Trp-mini cage protein using umbrella sampling MD simulations. Calculations were done with respect to increase in the temperature, and with respect to the concentration of the urea solution. The change in the free energy profiles, and the effect of external perturbations, and the atomistic details of the unfolding will be discussed. Temperature

Abstract

Design of biomimetic systems is an alternative method of finding biologically active molecules. Peptides incorporating tetrahydrofuran amino acids (TAA) adopt secondary folding patterns and thus mimic natural peptides. The preferred conformation of TAA derived linear and cyclized peptides depends on the stereochemistry of the tetrahydrofuran (THF) ring. Control of stereochemistry of THF ring in the structure of the linear tripeptide Boc-TAA-Leu-Val- OMe containing (2R,5S)-cis TAA, (2S,5R)-cis TAA, (2R,5R)-trans TAA and (2S,5S)-trans TAA is investigated using structural parameters and energetics data obtained from density functional theory (DFT) based calculations at B3LYP/6-31G(d,p) level of theory. We found that the conformations associated with -, -turn structures are stabilized by intramolecular hydrogen bonding interactions. Kinetic control of reactions leading to intra and inter molecular cyclization of tripeptide TAA-Gly-Gly containing (2S,5R)-cis TAA and (2S,5S)-trans TAA is studied at the same level of theory in gas phase.[1] We observe that kinetic control favors cyclodimerization instead of intramolecular cyclization.

Abstract

Macrocyclic trienes are important precursors for the synthesis of several biologically relevant molecules such as steroids. These molecules undergo (4+2) cycloaddition to form tricyclic compounds, which has four newly formed stereocenters. The stereochemistry at these four carbon centers has been shown to be largely controlled by the reaction energy barriers, and the conformational preferences of the macrocycle. Molecular dynamics (MD) simulation is a highly efficient approach for studying conformational properties of chemical molecules and biological macromolecules. MP2/6-31G* method was used to obtain potential energy surfaces corresponding to appropriate model compounds and used as the target data. The force field parameters were refined to accurately represent the target data involving all the model compounds chosen. Using the refined molecular mechanics force field, MD simulations were performed on six possible 14-membered symmetric trienes at different temperatures. We have also used replica exchange MD (RexMD) simulations with eight replicas between 300 and 370 K. The molecules were found to sample a larger conformational space in the RexMD simulation compared to the regular MD simulations. The probability distributions corresponding to the dihedral angles, and distances between the reaction centers have been analyzed. The relationship between the conformational sampling of the macrocyclic trienes vis-à-vis their reactivity will be discussed. Additionally, clustering of the structures from the MD trajectories was done to identify all unique conformations using a new approach.

Abstract

Sac7d is a protein present in Sulfolobus acidocaldarius, a hyperthermophilic archaeon, which is stable at extreme conditions. It is a chromosomal protein, which is capable of binding DNA thereby increasing its melting temperature. It was suggested to stabilize central 3 to 5 base pair steps in DNA. It binds to the minor groove of the DNA causing a kink in the helix without any sequential preference. We have used molecular dynamics (MD) simulations to study the binary complexes of Sac7d bound to DNA at 300 and 360 K. The objective is to understand the atomic detailed mechanism of the capability of Sac7d in facilitating DNA stability at higher temperature. As has been shown before, the structure of the protein is highly stable both at 300 and 360 K. The DNA duplex was found to be stable at 300 K and is similar to the X-ray crystal structure. Consistent with experiments, at least the central four base pairs of the DNA were found to be base paired during most of the simulation at 360 K. The dynamic properties of the protein in the protein-DNA complexes remained unchanged compared to when the protein is unbound. Interestingly, B- to A-form transitions occur in both the DNA molecules when bound to Sac7d, which was confirmed by sugar puckering angles, and backbone dihedral angles. Detailed examination of the protein-DNA interactions reveals that the stabilization of the DNA molecules is mostly due to the favorable interaction of the protein with the backbone of the DNA.

Abstract

Base flipping is a process by which one of the bases of the DNA swings out from its helical position to an extrahelical form. Several proteins such as methyltransferases, glycosylases and endonucleases employ this phenomenon in order to gain access to the target base for chemical modification. Even in the absence of protein, base pair opening occurs in nucleic acids in solution. X-ray crystal studies, fluorescence spectroscopy and NMR imino proton exchange experiments have been extensively employed to study the structures and dynamics involved in this fascinating event. We have employed molecular dynamics simulations to study base flipping both in presence and absence of the protein. The free energy pathways obtained using potential of mean force calculations will be presented. The base pair opening dynamics of AT and GC base pairs was investigated by calculating the equilibrium constants corresponding to the equilibrium between the base pair open and closed states, and compared with experimental data. Our calculations showed that the contribution to the overall equilibrium constants of the base pair opening is primarily from the purine bases. The methodological issues such as sufficient sampling and force field parameters will be addressed. In another study, the role of THR250 HhaI methyltransferase in base flipping will be discussed. THR250 is a highly conserved residue in methyltransferases and was proposed to be crucial for the correct placement of flipped cytosine for methylation. Previous computational studies have shown that the protein actively facilitates the base flipping process. Free energy calculations of base flipping in the presence of the wild type and T250G, T250A and T250D mutants of the protein were computed. The free energy profiles, structural changes and the energetic parameters for the base flipping process will be presented.

Abstract

Urea concentration dependent denaturation of proteins and nucleic acids has been widely used in obtaining (un)folding pathways and in calculating the free energies. Molecular dynamics simulations were performed on a 22-nt RNA hairpin in aqueous solution environment and in different concentrations (1-8M) of urea. The energetic and structural aspects of the role of urea in denaturating the RNA hairpin were investigated. In agreement with experimental studies, the RNA undergoes denaturation at higher concentrations of urea (> 6M). Surprisingly, the hairpin was observed to be more stable at lower concentrations of urea (1-2M) even compared to the RNA in aqueous environment. Various structural, energetic and solvation properties were analyzed to understand the mechanism of the interaction of urea with the hairpin. It was observed that urea does not directly facilitate the opening of the base pairs that leads to denaturation at higher concentrations. Instead, the open states of the RNA are stabilized by via hydrogen bonding interactions and stacking interactions thereby stabilizing the denatured form of the RNA.

Abstract

Transannular Diels-Alder reaction (TADA) combines traditional pericyclic and intramolecular reactions, and has been shown to be one of the most efficient chemical transformations for synthesizing complicated natural products. TADA reactions occur in (x+y+2)-membered triene macrocycles, which contain both the diene and the dienophile, to form A.B.C[x.6.y] type tricyclic compounds. In addition, these macrocycles were shown to interconvert among its isomers via [1,5]-sigmatropic hydrogen shifts. The stereochemical outcome of the TADA reactions depends on the energetics of various possible transition states. Initially, a validation study was undertaken to assess the performance of various ab initio (HF, MPx, and CC) and hybrid density functional B3LYP methods using a variety of basis sets. Comparison of the reaction barriers, and reaction energies from various methods indicate that B3LYP/6-31G* level is appropriate enough for accurately modeling this class of reactions. All possible reaction pathways involving the TADA and 1,5-sigmatropic reactions of six different 14-membered triene molecules were studied using B3LYP/cc-pVTZ were calculated. Based on the reaction energy barriers and conformational properties of the reactants, the reactivities of these molecules will be discussed.

Abstract

Proteins, which are the most common drug targets, are flexible and can exist in various conformers that are energetically competitive. Hence, it is crucial to consider its dynamic behavior. Most notable methods that incorporate flexibility of protein are discussed here. Structural knowledge of molecular targets (or drug receptors), usually proteins, has transformed the drug design process in the last three decades or so. While most of the drugs that were discovered 30 years ago are by serendipity and ‘trial and error’, rational drug design approaches are more focused. Rational drug design techniques use structure of the drug receptor or structure of ligands that are shown to bind to the target to identify potential drug candidates. Approaches that use the structure of drug receptor for drug discovery are referred to as structure based drug design (SBDD). X-ray crystallography and NMR spectroscopy have been in the forefront in protein structure determination. Several computational methods have been developed and new methods are being proposed for protein structure prediction from the primary sequence (for eg. homology modeling); however, they suffer from several caveats. Currently, use of SBDD has become a standard exercise as part of drug discovery and development both in industry and academia. Typically, the process involves obtaining the structure of the target protein; identification of active site; virtual screening of a small molecule database (containing chemically diverse structures of small molecules in the order of a million) and identification of potential ligands based on a chosen scoring function. Ideally, docking process involved in virtual screening and calculating the binding energy should allow the protein and the ligand to change their conformations to effectively bind to each other. Ligands being smaller compounds (typically containing tens of nonhydrogen atoms), their conformational changes can be modeled with reasonable accuracy. However, explicit modeling of protein flexibility involves a lot of computer time especially considering that a million compounds have to be screened and hence is not feasible. In short, a compromise between ‘quick answer’ and ‘right answer’ is made in docking calculations for practical applications. Recently, allowing protein to be flexible at least partially has become possible, thanks to development of efficient algorithms and, availability of faster processors, larger storage and RAM. Several approximations have been proposed to accommodate protein flexibility of which few of the notable ones are discussed in the article. The readers are suggested to refer additional resources on structure based drug design, which are given at the end.

Abstract

Proteins from hyperthermophilic microorganisms possess unique structurefunction properties for surviving high temperatures and for exhibiting optimal activity. In an attempt to understand the structural and energetic factors that are responsible for the thermal stability, molecular dynamics (MD) simulations were performed on Sso7d and Sac7d chromosomal proteins from S. solfataricus and S. acidocaldarius respectively. Consistent with experimental data, the proteins are quite stable at 300 and 360 K, but were found to undergo denaturation at higher temperature. Both proteins exhibit similar hierarchical unfolding pathways, which is explained based on the calculated intramolecular interaction energies. Differential dynamic behaviors of these proteins were examined, and possible roles of enzymatic activity of Sso7d are proposed. Structural, energetic, dynamic and solvation properties that influence the stability of these proteins will be discussed. The importance of hydrophobic interactions on their thermal stability will be presented using calculations on a mutant (F31A in Sso7d). These proteins are also capable of binding to DNA in a nonspecific fashion, and upon binding, melting temperature of the DNA was found to increase by about 40 K. MD simulations were also performed on protein-DNA complexes to investigate the stability of the oligonucleotide. Possible factors that facilitate the stability of these binary complexes in extraordinary thermal conditions will be discussed based on intra- and intermolecular interaction energies, correlated movements between the protein and DNA, solvation properties, and other structural parameters.

Abstract

Computational approaches are being successfully utilized for the design of small molecules that could be potential substrates for various biological targets both in academia and pharmaceutical industry. Over the past two decades or so, various computational techniques have been developed and improvements on the existing methodologies were proposed to accomplish high accuracy/ predictability of molecular properties. Consideration of the flexible nature of proteins in structure based drug design studies has been a bottleneck mainly due to the huge demand of computational and actual time. Hence most of the docking studies consider the biological macromolecules to be rigid and allow only the conformational changes of the small molecules. Similarly, the ligand based drug design approaches tend to focus more on the global minima of the small molecules. This could lead to incorrect model and hence false predictions since this approximation may not be true in cases where the global minima essentially is not the bioactive conformer – the conformer that binds to the target. Recently, advanced methods have been proposed to consider the flexible nature of the molecules. Molecular dynamics (MD) simulations have been shown to be a straightforward means to include the flexibility of both ligands and biomolecular targets. MD simulations are invaluable computational methods that are extensively used for deriving information of the structure, dynamics and energetic properties of various biomolecules including proteins, nucleic acids and lipids. The need for consideration of dynamic nature of the biomolecular targets will be demonstrated and the advantages of MD simulations to overcome this limitation will be discussed. Basic principles of MD simulations will be discussed and an introduction to biomolecular force fields will be given. Illustrated examples of the applications of MD simulations to ligand based and structure based drug design approaches will be presented.