The mechanism of the converter domain rotation in the recovery stroke of myosin motor protein

Upon ATP binding, myosin motor protein is found in two alternative conformations, prerecovery state M* and postrecovery state M**. The transition from one state to the other, known as the recovery stroke, plays a key role in the myosin functional cycle. Despite much recent research, the microscopic details of this transition remain elusive. A critical step in the recovery stroke is the rotation of the converter domain from “up” position in prerecovery state to “down” position in postrecovery state that leads to the swing of the lever arm attached to it. In this work, we demonstrate that the two rotational states of the converter domain are determined by the interactions within a small structural motif in the force‐generating region of the protein that can be accurately modeled on computers using atomic representation and explicit solvent. Our simulations show that the transition between the two states is controlled by a small helix (SH1) located next to the relay helix and relay loop. A small translation in the position of SH1 away from the relay helix is seen to trigger the transition from “up” state to “down” state. The transition is driven by a cluster of hydrophobic residues I687, F487, and F506 that make significant contributions to the stability of both states. The proposed mechanism agrees well with the available structural and mutational studies. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Interactions between relay helix and Src homology 1 (SH1) domain helix drive the converter domain rotation during the recovery stroke of myosin II

Myosin motor protein exists in two alternative conformations, prerecovery state M* and postrecovery state M**, on adenosine triphosphate binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the postrecovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this article, we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain, and Src homology 1 (SH1) domain helix, with which relay helix interacts and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models, we find that: (1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein and (2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix, which is subsequently transmitted to the converter domain.     

Understanding the folding and stability of a designed WW domain protein with replica exchange molecular dynamics simulations

WW domain proteins are usually regarded as simple models for understanding the folding mechanism of β-sheet. CC45 is an artificial protein that is capable of folding into the same structure as WW domain. In this article, the replica exchange molecular dynamics simulations are performed to investigate the folding mechanism of CC45. The analysis of thermal stability shows that β-hairpin 1 is more stable than β-hairpin 2 during the unfolding process. Free energy analysis shows that the unfolding of this protein substantially proceeds through solvating the smaller β-hairpin 2, followed by the unfolding of β-hairpin 1. We further propose the unfolding process of CC45 and the folding mechanism of two β-hairpins. These results are similar to the previous folding studies of formin binding protein 28 (FBP28). Compared with FBP28, it is found that CC45 has more aromatic residues in N-terminal loop, and these residues contact with C-terminal loop to form the outer hydrophobic core, which increases the stability of CC45. Knowledge about the stability and folding behaviour of CC45 may help in understanding the folding mechanisms of the β-sheet and in designing new WW domains.     

Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.     

Structure-function analysis suggests that the photoreceptor LITE-1 is a light-activated ion channel

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Understanding the folding and stability of a zinc finger-based full sequence design protein with replica exchange molecular dynamics simulations

Full sequence design protein FSD-1 is a designed protein based on the motif of zinc finger protein. In this work, its folding mechanism and thermal stability are investigated using the replica exchange molecular dynamics model with the water molecules being treated explicitly. The results show that the folding of the FSD-1 is initiated by the hydrophobic collapse, which is accompanied with the formation of the C-terminal alpha-helix. Then the folding proceeds with the formation of the beta-hairpin and the further package of the hydrophobic core. Compared with the beta-hairpin, the alpha-helix has much higher stability. It is also found that the N-capping motif adopted by the FSD-1 contributes to the stability of the alpha-helix dramatically. The hydrophobic contacts made by the side chain of Tyr3 in the native state are essential for the stabilization of the beta-hairpin. It is also found that the folding of the N-terminal beta-hairpin and the C-terminal alpha-helix exhibits weak cooperativity, which is consistent with the experimental data. Meanwhile, the folding pathway is compared between the FSD-1 and the target zinc finger peptide, and the possible role of the zinc ion on the folding pathway of zinc finger is proposed. Proteins 2007. (c) 2007 Wiley-Liss, Inc.     

Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.

The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics studies of the antimicrobial peptides piscidin 1 and its mutants with a DOPC lipid bilayer

Piscidin 1 (Pis‐1) has a high broad‐spectrum activity against bacteria, fungi, and viruses but it also has a moderate hemolytic activities. To improve the antibacterial activity and to reduce toxicity, mutants Pis‐1AA (G8A/G13A double mutant) and Pis‐1PG (G8P mutant) have been designed based on the crystal structure of Pis‐1. Eighteen independent molecular dynamics (MD) simulations of Pis‐1 and its mutants with membranes are conducted in this article. Furthermore, 60 independent MD simulations of three peptides in water box have also been discussed for comparison. The results indicate that the unfolding process starts at the middle of the peptide. Pis‐1 disrupts easily in the region of Val10‐Lys14. Pis‐1PG has a flexible N‐terminal region, and the interaction between N‐terminal and C‐terminal is very weak. Pis‐1AA has the most stable helical structure. In addition, percentage of native contacts and hydrogen bonds analysis are also performed. Lipid‐peptide interaction analysis suggests that Pis‐1 and Pis‐1AA has a stronger interaction with the zwitterionic dioleoylphosphatidylcholine (DOPC) lipid bilayer than Pis‐1PG. When compared with the results of peptide with membrane, peptides are unstable and unfolding quickly in water solution. Our results are applicable in examining diversities on hemolytic, antibacterial, and selectivity of antimicrobial peptides. © 2012 Wiley Periodicals, Inc. Biopolymers 97:998–1009, 2012.     

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α‐Crystallin is a multimeric eye lens protein having molecular chaperone‐like function which is crucial for lens transparency. The stability and unfolding‐refolding properties of α‐crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native‐like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α‐crystallin revealed dry native‐like core of α‐crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol^?1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol^?1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014.     

Sequence-dependent dynamical instability of the human prion protein: a comparative simulation study

The present study aimed to explore the most probable regions of the human prion protein backbone for which the initial steps of conformational transitions as a result of intrinsic and extrinsic perturbing factors on the protein structure can be assigned. A total of 0.3-μs molecular dynamics simulations on several analog structures of the protein have been performed. To mimic the impact of the extrinsic and intrinsic destructive parameters on the dynamical characteristics of the protein, mild acidic conditions and R208H mutation have been simulated. The findings indicated that distribution of conformational flexibilities along the protein chain was almost independent of the induced perturbing factors, and was mostly centralized on certain distinct parts of the structure comprising residues 132–145 and 187–203. Analyses also revealed that the segment comprising residues 187–203 may be considered as a peptide sequence, possessing high potential to start the initial steps of conformational rearrangements due to the induced physicochemical alterations. Sequence alignment and molecular dynamics data also revealed that segment 178–203 prefers to accommodate in extended structures rather than α-helices. Region 178–203 may be considered as a peptide switch capable of initiating the conformational transitions due to the introduced modifications and perturbing parameters.     

Investigation on bioactive protection of the amino acids derived from LEA protein on insulin by molecular simulation

Nowadays heat-sensitive protein medicines are increasingly showing their importance in the treatment of various diseases. Their popularisation and application are meeting a great challenge because of their heat lability. In this study, human insulin as a heat-sensitive protein medicine and 66 amino acids derived from a Group 3 late embryogenesis abundant protein fragment as a complex bioactive protectant, were chosen to be investigated to determine whether these amino acids can be used to protect the insulin from denaturation due to drying. The experiments were carried out by using a replica exchange molecular dynamics (REMD) simulation and GROMACS software with Gromos96 (53a6) force field. The REMD results indicate that those amino acids can effectively prevent the reversal between hydrophilic and hydrophobic surface. Both the configurations and secondary structures of the protected insulin were preserved very well. The H-bonding and electrostatic interactions between the insulin and the protectant play key roles in the bioactive protection of insulin. These results agree well with the water replacement hypothesis. All the results prove that these amino acids are a perfect bioactive protectant for heat-sensitive protein medicines.     

Free energy landscape and folding mechanism of a beta-hairpin in explicit water: a replica exchange molecular dynamics study

The free energy landscape and the folding mechanism of the C-terminal beta-hairpin of protein G is studied by extensive replica exchange molecular dynamics simulations (40 replicas and 340 ns total simulation time), using the GROMOS96 force field and the SPC explicit water solvent. The study reveals that the system preferentially adopts a beta-hairpin structure at biologically important temperatures, and that the helix content is low at all temperatures studied. Representing the free energy landscape as a function of several types of reaction coordinates, four local minima corresponding to the folded, partially folded, molten globule, and unfolded states are identified. The findings suggest that the folding of the beta-hairpin occurs as the sequence: collapse of hydrophobic core --> formation of H-bond --> formation of the turn. Identifying the folded and molten globule states as the main conformations, the free energy landscape of the beta-hairpin is consistent with a two-state behavior with a broad transition state. The temperature dependence of the folding-unfolding transition is investigated in some detail. The enthalpy and entropy jumps at the folding transition temperature are found to be about three times lower than the experimental estimates, indicating that the folding-unfolding transition in silico is less cooperative than its in vitro counterpart.     

Mimicking the action of folding chaperones by Hamiltonian replica-exchange molecular dynamics simulations: application in the refinement of de novo models

The efficiency of using a variant of Hamiltonian replica‐exchange molecular dynamics (Chaperone H‐replica‐exchange molecular dynamics [CH‐REMD]) for the refinement of protein structural models generated de novo is investigated. In CH‐REMD, the interaction between the protein and its environment, specifically, the electrostatic interaction between the protein and the solvating water, is varied leading to cycles of partial unfolding and refolding mimicking some aspects of folding chaperones. In 10 of the 15 cases examined, the CH‐REMD approach sampled structures in which the root‐mean‐square deviation (RMSD) of secondary structure elements (SSE‐RMSD) with respect to the experimental structure was more than 1.0 Å lower than the initial de novo model. In 14 of the 15 cases, the improvement was more than 0.5 Å. The ability of three different statistical potentials to identify near‐native conformations was also examined. Little correlation between the SSE‐RMSD of the sampled structures with respect to the experimental structure and any of the scoring functions tested was found. The most effective scoring function tested was the DFIRE potential. Using the DFIRE potential, the SSE‐RMSD of the best scoring structures was on average 0.3 Å lower than the initial model. Overall the work demonstrates that targeted enhanced‐sampling techniques such as CH‐REMD can lead to the systematic refinement of protein structural models generated de novo but that improved potentials for the identification of near‐native structures are still needed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Salt effects on hydrophobic‐core formation in folding of a helical miniprotein studied by molecular dynamics simulations

We have investigated effects of salt ions on folding events of a helical miniprotein chicken villin headpiece subdomain HP36. Low concentrations of ions alter electrostatic interactions between charged groups of a protein and can change the populations of conformers. Here, we compare two data sets of folding simulations of HP36 in explicit water solvent with or without ions. For efficient sampling of the conformational space of HP36, the multicanonical replica‐exchange molecular dynamics method was employed. Our analyses suggest that salt alters salt‐bridging nature of the protein at later stages of folding at room temperature. Especially, more nonnative, nonlocal salt bridges are formed at near‐native conformations in pure water. Our analyses also show that such salt‐bridge formation hinders the fully native hydrophobic‐core packing at the final stages of folding. Proteins 2014; 82:933–943. © 2013 Wiley Periodicals, Inc.     

Study of the impact of the T877A mutation on ligand‐induced helix‐12 positioning of the androgen receptor resulted in design and synthesis of novel antiandrogens

Antiandrogen flutamide, an antagonist of the wild‐type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist‐agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand‐binding domain (AR‐LBD), we have investigated the impact of the T877A mutation on ligand‐induced helix‐12 positioning by replica‐exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR‐LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild‐type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non‐agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan‐antiandrogen effective against the wild‐type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Molecular dynamics simulation and experimental validation of the effect of pH on protein desorption in hydrophobic charge induction chromatography

The ligands in hydrophobic charge induction chromatography (HCIC) are hydrophobic and ionisable. Thus, the pH is crucial for the separation performance in HCIC, especially for elution. However, it is difficult to obtain the microscopic information in HCIC through experimental means. In this work, molecular dynamics simulations are performed to examine the effect of pH on elution and protein conformational transition in HCIC, using a 46-bead β-barrel coarse-grained model protein and an HCIC adsorbent pore model constructed in an earlier work. Corresponding experiments are carried out for the validation of simulation results, using lysozyme and MEP Hypercel. Both the activities and fluorescence of lysozyme are examined to evaluate the conformational transition. The simulations indicate that the elution efficiency of protein increases with decreasing pH value in a non-linear manner. This is qualitatively consistent with the experimental results. MD simulations indicate that protein unfolding occurs in elution at all pH values. However, the experimental data show that the activity and conformation of lysozyme is independent of pH of the elution buffer. The microscopic information from simulation shows that protein unfolding is mainly observed on the adsorbent surface, but it cannot be detected in the experiments that only probe the proteins in the bulk liquid.     

Protein dynamics in a family of laboratory evolved thermophilic enzymes

Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm(-1)), with the more stable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.     

Protein recycling is a major component of post-irradiation recovery in Deinococcus radiodurans strain R1

Exposure to 6kGy dose of (60)Co gamma-rays resulted in immediate growth arrest, followed by complete recovery of Deinococcus radiodurans strain R1 cells. Selective degradation and resynthesis of several predicted highly expressed proteins (including major chaperones, key TCA cycle enzymes, and few stress proteins) and several hypothetical proteins marked the lag period, preceding resumption of growth. A major exercise in protein recycling appears to be an integral component of post-irradiation recovery in D. radiodurans and complements the extensive DNA repair, characteristic of this extremely radioresistant bacterium.     

Two enzymatic reaction pathways in the formation of pyropheophorbide a

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated `pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13<sup>2</sup>-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed `pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K _m values against pheophorbide a for both Phedases were 14–15 μM and 283 μM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of charge redistribution on the binding of DNA nucleotide to carbon nanotube in molecular dynamics

All-atom molecular dynamics (MD) simulations are performed to study the binding of DNA nucleotides with two carbon nanotubes (CNTs) with similar diameters but different chiralities. Two schemes for assigning partial atomic charges (PACs) are adopted: (I) using PACs obtained from isolated DNA nucleotide and CNT optimised in vacuum, and (II) using PACs obtained from optimising nucleotide-CNT hybrid in solution. The former approach is what most MD simulations have used in the study of DNA-CNT hybrids, while in the latter approach, a redistribution of the PACs has occurred upon the hybridisation. Our results show that the charge redistribution has a profound effect on the dynamics of binding. In particular, PACs obtained from (II) lead to more stable binding structures in the MD simulations. The findings suggest that care should be taken in simulating DNA-CNT interactions using the classical force field approach.