Methanol Concentration Dependent Protein Denaturing Ability of Guanidinium/Methanol Mixed Solution

Mixtures of osmolytes are present in the cell. Therefore, the understanding of the interplay of mixed osmolyte molecules and their combined effects on protein structure is of fundamental importance. In this article, the structure stability of a model protein (BdpA) in the mixture of guanidinium thiocyanate (GdmSCN) and methanol (MeOH) was investigated by molecular dynamics simulation. It was observed that guanidinium (Gdm⁺) is driven to protein surface by favorable electrostatic interactions and MeOH is driven by both favorable electrostatic and VDW interactions, respectively. The mixture of Gdm⁺ and MeOH doesnot affect the electrostatic energy distribution of Gdm⁺ but does reduce the difference in VDW energy of MeOH between the regions of protein surface and bulk solution. As a result, the accumulation level of Gdm⁺ is not influenced, but the accumulation level of MeOH is lowered in mixed solution. The tertiary structure stability of protein is determined by the accumulated strength of VDW interactions from MeOH to protein side chain, and the secondary structure stability is correlated to the strength of combined electrostatic energies from solvent (water) and cosolvent (Gdm⁺ and MeOH) to protein backbone, particularly in hydrogen bonding part. The mixture of GdmSCN with low-concentrated MeOH stabilizes native structure of BdpA whereas the further increase of MeOH concentration denatures native structure of protein to expanded unfolded structure. The present study together with our previous study on the mixture of GdmSCN and 2,2,2-trifluoroethanol (TFE) provides novel insights into the effects of mixed osmolytes on protein structure

Folding or Misfolding: The Choice of β‑Hairpin

Proteins fold through complex inter-residue interactions which are mutually supportive and cooperatively lead to thermodynamically favorable native structures. Competing (misfolded) structures, however, could exist, which might affect the thermodynamic and kinetic properties of folded structure. Running long-time REMD simulations on two β-structured polypeptides, the present study identifies the folded and (less populated) competing misfolded states of β-hairpins. Of particular interest is a one-residue shifted misfolded state which has been often seen in previous reports. The folding and misfolding pathways are then energetically characterized by free energy landscape analysis, indicating that the folding and misfolding of β-hairpin are parallel pathways and a protein’s selection of following which pathway is a consequence of the competition between the formation of alterable turn configurations and cross-strand hydrophobic interactions. Proteins possessing high percentage of hydrophobic residues introduce strong cross-strand hydrophobic interactions which stabilize the native structural elements in the folding pathway, leading to low possibility of misfolding. The present study provides novel insights into the origin of sequence-dependent β-hairpin misfolding “hidden” behind experimentally detectable β-hairpin folding, suggesting the direction for the structure design of β-structured protein

Probing Sequence Dependence of Folding Pathway of α‑Helix Bundle Proteins through Free Energy Landscape Analysis

A comparative study on the folding of multiple three-α-helix bundle proteins including α₃D, α₃W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation–condensation mechanism (e.g., α₃D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α₃W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins

How Well Can Implicit Solvent Simulations Explore Folding Pathways? A Quantitative Analysis of α‑Helix Bundle Proteins

Protein folding has been posing challenges for molecular simulation for decades. Implicit solvent models are sought as routes to increase the capability of simulation, with trade-offs between computational speed and accuracy. Here, we systematically investigate the folding of a variety of α-helix bundle proteins ranging in size from 46 to 102 amino acids using a state-of-the-art force field and an implicit solvent model. The accurate all-atom simulated folding is enabled for six proteins, including for the first time a successful folding of protein with >100 amino acids in implicit solvent. The detailed free-energy landscape analysis sheds light on a set of general principles underlying the folding of α-helix bundle proteins, suggesting a hybrid framework/nucleation-condensation mechanism favorably adopted in implicit solvent condition. The similarities and discrepancies of the folding pathways measured among the present implicit solvent simulations and previously reported experiments and explicit solvent simulations are deeply analyzed, providing quantitative assessment for the availability and limitation of implicit solvent simulation in exploring the folding transition of large-size proteins

Effective Conformational Sampling in Explicit Solvent with Gaussian Biased Accelerated Molecular Dynamics

In this Article, a user-friendly Gaussian biased accelerated molecular dynamics (GbAMD) method is presented that uses a sum of Gaussians of potential energies as the biased force to accelerate the conformational sampling. The easy parameter setting of GbAMD is demonstrated in a variety of simulation tests for the conformational transitions of proteins with various complexity including the folding of Trpcage, GB1p, and HP35 peptides as well as the functional conformational changes of nCaM and HIV-1 PR proteins. Additionally, the ability of GbAMD in conformational sampling and free-energy evaluation is quantitatively assessed through the comparison of GbAMD simulations on the folding of α-helical Trpcage and β-hairpin GB1p with the accompanying standard dual boost AMD and conventional MD (cMD) simulations. While GbAMD can fold both peptides into their native structures repeatedly in individual trajectories, AMD can only fold Trpcage and cMD fails the folding in both cases. As a result, only GbAMD can quantitatively measure the properties of the equilibrium conformational ensemble of protein folding consistent with experimental data. Also notable is that the structural properties of the indispensable unfolded and transition states in the folding pathways of Trpcage and GB1p characterized by GbAMD simulations are in great agreement with previous simulations on the two peptides. In summary, GbAMD has an effective conformational sampling ability that provides a convenient and effective access for simulating the structural dynamics of biomolecular systems

Determining Protein Folding Pathway and Associated Energetics through Partitioned Integrated-Tempering-Sampling Simulation

Replica exchange molecular dynamics (REMD) and integrated-tempering-sampling (ITS) are two representative enhanced sampling methods which utilize parallel and integrated tempering approaches, respectively. In this work, a partitioned integrated-tempering-sampling (P-ITS) method is proposed which takes advantage of the benefits of both parallel and integrated tempering approaches. Using P-ITS, the folding pathways of a series of proteins with diverse native structures are explored on multidimensional free-energy landscapes, and the associated thermodynamics are evaluated. In comparison to the original form of ITS, P-ITS improves the sampling efficiency and measures the folding/unfolding thermodynamic quantities more consistently with experimental data. In comparison to REMD, P-ITS significantly reduces the requirement of computational resources and meanwhile achieves similar simulation results. The observed structural characterizations of transition and intermediate states of the proteins under study are in good agreement with previous experimental and simulation studies on the same proteins and homologues. Therefore, the P-ITS method has great potential in simulating the structural dynamics of complex biomolecular systems

Specific Inhibitory Effect of κ-Carrageenan Polysaccharide on Swine Pandemic 2009 H1N1 Influenza Virus

<div><p>The 2009 influenza A H1N1 pandemic placed unprecedented demands on antiviral drug resources and the vaccine industry. Carrageenan, an extractive of red algae, has been proven to inhibit infection and multiplication of various enveloped viruses. The aim of this study was to examine the ability of κ-carrageenan to inhibit swine pandemic 2009 H1N1 influenza virus to gain an understanding of antiviral ability of κ-carrageenan. It was here demonstrated that κ-carrageenan had no cytotoxicity at concentrations below 1000 μg/ml. Hemagglutination, 50% tissue culture infectious dose (TCID₅₀) and cytopathic effect (CPE) inhibition assays showed that κ-carrageenan inhibited A/Swine/Shandong/731/2009 H1N1 (SW731) and A/California/04/2009 H1N1 (CA04) replication in a dose-dependent fashion. Mechanism studies show that the inhibition of SW731 multiplication and mRNA expression was maximized when κ-carrageenan was added before or during adsorption. The result of Hemagglutination inhibition assay indicate that κ-carrageenan specifically targeted HA of SW731 and CA04, both of which are pandemic H1N/2009 viruses, without effect on A/Pureto Rico/8/34 H1N1 (PR8), A/WSN/1933 H1N1 (WSN), A/Swine/Beijing/26/2008 H1N1 (SW26), A/Chicken/Shandong/LY/2008 H9N2 (LY08), and A/Chicken/Shandong/ZB/2007 H9N2 (ZB07) viruses. Immunofluorescence assay and Western blot showed that κ-carrageenan also inhibited SW731 protein expression after its internalization into cells. These results suggest that κ-carrageenan can significantly inhibit SW731 replication by interfering with a few replication steps in the SW731 life cycles, including adsorption, transcription, and viral protein expression, especially interactions between HA and cells. In this way, κ-carrageenan might be a suitable alternative approach to therapy meant to address anti-IAV, which contains an HA homologous to that of SW731.</p></div

Counterion Effects on the Denaturing Activity of Guanidinium Cation to Protein

The denaturation of a three-α-helix bundle, the B domain of protein A, by guanidinium is studied by molecular dynamics simulations. The simulation results showed that in GdmCl solution, guanidinium cations accumulate around the protein surface, whereas chloride anions are expelled from the protein. In contrast, in GdmSCN solution, both cations and anions accumulate around the protein surface and the degree of Gdm⁺ accumulation is higher than that in GdmCl, suggesting the cooperativity between the cations and anions in preferential binding. Moreover, the accumulation of guanidinium around the protein surface is not uniform, and it prefers to populate near residues with negatively charged or planar side chains. On the other hand, guanidinium participates in direct hydrogen bonding with backbone carbonyl groups. Meanwhile, guanidinium also promotes the hydrogen bonding of water to a backbone carbonyl group by changing the hydrogen bonding network within solvent. Therefore, the attack from both water and guanidinium breaks backbone hydrogen bonds and results in the destruction of secondary structures of the protein. The stronger accumulation of guanidinium and more hydrogen bonding from guanidinium in GdmSCN leads to the increase of its denaturing efficiency compared to GdmCl. In the latter solution, the ion pairing between Cl^– and guanidinium limits the approach of guanidinium to protein and the hydrogen bonding between guanidinium and protein, and the main denaturing contributor is the hydrogen bonding from water

Anti-SW731 effect of κ-carrageenan on MDCK cells.

<p>(A, B) MDCK cells were incubated with SW731, CA04, PR8, WSN, and ZB07 at 4°C (MOI = 0.1) and then treated with κ-carrageenan or Ribavirin at the indicated concentration after removal of the virus inoculum. After 24 h, the viral titers were determined by HA and TCID50 assays. (C, D) MDCK cells were incubated with SW731, CA04, PR8, WSN, and ZB07 at 4°C (MOI = 1) and then treated with κ-carrageenan or Ribavirin at the indicated concentration after removal of the virus inoculum. After 24 h, the viral titers were determined by HA and TCID₅₀ assays. (E) MDCK cells were infected with SW731 at an MOI of 1 and then treated with κ-carrageenan at the indicated concentration after removal of the viral inoculum. After 48 h, CPE inhibition was determined by CPE assay. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *<i>P <</i> 0.05 vs. nondrug treated control group; **<i>P <</i> 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.</p

Cytotoxic effect of κ-carrageenan on MDCK cells.

<p>MDCK cells were treated with the indicated concentrations of κ-carrageenan. After 48 h of incubation, metabolic activity was measured via MTT assay. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM (n = 3). Significance: *<i>P <</i> 0.05 vs. nondrug-treated control group; **<i>P <</i> 0.005 vs. nondrug-treated control group. Results are representative of two independent experiments.</p