Investigations of the thermostability of rubredoxin models using molecular dynamics simulations.

The affects of differences in amino acid sequence on the temperature stability of the three-dimensional structure of the small beta-sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100 degrees C, were compared to three mesophilic Rds of known X-ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature-dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature-dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.     

Structural stability of disulfide mutants of basic pancreatic trypsin inhibitor: A molecular dynamics study

The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5–55] and [30–51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or folding processes. © 1996 Wiley-Liss, Inc.     

Heterogeneous folding of the trpzip hairpin: full atom simulation and experiment

The beta-hairpin trpzip2 can be tuned continuously from a two-state folder to folding on a rough energy landscape without a dominant refolding barrier. At high denaturant concentration, this extremely stable peptide exhibits a single apparent "two-state" transition temperature when monitored by different spectroscopic techniques. However, under optimal folding conditions the hairpin undergoes an unusual folding process with three clusters of melting transitions ranging from 15 degrees C to 160 degrees C, as monitored by 12 different experimental and computational observables. We explain this behavior in terms of a rough free energy landscape of the unfolded peptide caused by multiple tryptophan interactions and alternative backbone conformations. The landscape is mapped out by potentials of mean force derived from replica-exchange molecular dynamics simulations. Implications for deducing cooperativity from denaturant titrations, for the origin of folding cooperativity, and for the folding of thermophilic proteins are pointed out. trpzip is an excellent small tunable model system for the glass-like folding transitions predicted by landscape theory.     

Repetitive pulling catalyzes co-translocational unfolding of barnase during import through a mitochondrial pore

We present a computational study of barnase unfolding during import into mitochondria through a model translocon. In contrast to thermal (or chemical) unfolding, the major intermediates of co-translocational unfolding are mainly mediated by non-native interactions accompanying the protein configurations induced by pulling forces. These energy contributions, combined with backbone topological constraints imposed by the model pore, result in milestones along the unfolding pathways which are significantly different not only from those experienced during thermal (or chemical) denaturation, but also from those observed in single-molecule pulling by both ends without pore constraints. Two on-pathway major translocation intermediates trapped in long-lived states by significantly high unfolding barriers are identified. A fraction of these pathways can, however, skip such local kinetic traps and result in extremely fast translocations, leading to a dramatic kinetic partitioning spanning approximately four orders of magnitude. The fraction of fast translocation events is shown to increase upon switching the pull off and on, when compared to pulling at constant force. This suggests a "catalytic" mechanism by which the mitochondrial import machinery regulates this partitioning by repetitively pulling in cycles. A number of mutation sites that alter the kinetic "flow" of the unfolding trajectories are suggested and tested.     

Unfolding a linker between helical repeats

In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for alpha-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins.     

A study of the structural properties and thermal stability of human Bcl-2 by molecular dynamics simulations

The anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein interacts with several proteins that regulate the apoptotic properties of cells. In this research, we conduct several all-atom molecular dynamics (MD) simulations under high-temperature unfolding conditions, from 400 to 800?K, for 25?ns. These simulations were performed using a model of an engineered Bcl-2 human protein (Bcl-2-Δ22Σ3), which lacks 22 C-terminal residues of the transmembrane domain. The aim of this study is to gain insight into the structural behavior of Bcl-2-Δ22Σ3 by mapping the conformational movements involved in Bcl-2 stability and its biological function. To build a Bcl-2-Δ22Σ3 three-dimensional model, the protein core was built by homology modeling and the flexible loop domain (FLD, residues 33-91) by ab initio methods. Further, the entire protein model was refined by MD simulations. Afterwards, the production MD simulations showed that the FLD at 400 and 500?K has several conformations reaching into the protein core, whereas at 600?K some of the alpha-helices were lost. At 800?K, the Bcl-2 core is destabilized suggesting a possible mechanism for protein unfolding, where the alpha helices 1 and 6 were the most stable, and a reduction in the number of hydrogen bonds initially occurs. In conclusion, the structural changes and the internal protein interactions suggest that the core and the FLD are crucial components of Bcl-2 in its function of regulate ng access to the recognition sites of kinases and caspases.     

Mutational analysis of FUS gene and its structural and functional role in amyotrophic lateral sclerosis 6

Amyotrophic lateral sclerosis 6 (ALS6) is an autosomal recessive disorder caused by heterozygous mutation in the Fused in Sarcoma (FUS) gene. ALS6 is a neurodegenerative disorder, which affects the upper and lower motor neurons in the brain and spinal cord, resulting in fatal paralysis. ALS6 is caused by the genetic mutation in the proline/tyrosine-nuclear localization signals of the Fused in sarcoma Protein (FUS). FUS gene also known as TLS (Translocated in liposarcoma), which encodes a protein called RNA-binding protein-Fus (FUS), has a molecular weight of 75?kDa. In this analysis, we applied computational approach to filter the most deleterious and neurodegenerative disease of ALS6-associated mutation on FUS protein. We found H517Q as most deleterious and disease associated using PolyPhen 2.0, I-Mutant 3.0, SIFT, SNPs&GO, PhD-SNP, Pmut, and Mutpred tools. Molecular dynamics simulation (MDS) approach was conducted to investigate conformational changes in the mutant protein structure with respect to its native conformation. MDS results showed the flexibility loss in mutant (H517Q) FUS protein. Due to mutation, FUS protein became more rigid in nature and might alter the structural and functional behavior of protein and play a major role in inducing ALS6. The results obtained from this investigation would help in the field of pharmacogenomics to develop a potent drug target against FUS-associated neurodegenerative diseases.