Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Molecular dynamics simulations of the unfolding of an alpha-helical analogue of ribonuclease A S-peptide in water

Molecular dynamics simulations of the S-peptide analogue AETAAAKFLREHMDS have been conducted in aqueous solution for 300 ps at 278 K and for 500 ps in two different runs at 358 K. The results show agreement with experimental observations in that at low temperature, 5 degrees C, the helix is stable, while unfolding is observed at 85 degrees C. In the low-temperature simulation a solvent-separated ion pair was formed between Glu-2 and Arg-10, and the side chain of His-12 reoriented toward the C-terminal end of the alpha-helix. Detailed analyses of the unfolding pathways at high temperature have also revealed that the formation or disappearance of main-chain helical hydrogen bonds occurs frequently through an alpha in equilibrium with 3(10) in equilibrium with no hydrogen bond sequence.     

Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

Molecular dynamics simulation study on the structures of fascin mutants

Fascin is a filamentous actin (F-actin) bundling protein, which cross-links F-actin into bundles and becomes an important component of filopodia on the cell surface. Fascin is overexpressed in many types of cancers. The mutation of fascin affects its ability to bind to F-actin and the progress of cancer. In this paper, we have studied the effects of residues of K22, K41, K43, K241, K358, K399, and K471 using molecular dynamics (MD) simulation. For the strong-effect residues, that is, K22, K41, K43, K358, and K471, our results show that the mutation of K to A leads to large values of root mean square fluctuation (RMSF) around the mutated residues, indicating those residues are important for the flexibility and thermal stability. On the other hand, based on residue cross-correlation analysis, alanine mutations of these residues reinforce the correlation between residues. Together with the RMSF data, the local flexibility is extended to the entire protein by the strong correlations to influence the dynamics and function of fascin. By contrast, for the mutants of K241A and K399A those do not affect the function of fascin, the RMSF data do not show significant differences compared with wild-type fascin. These findings are in a good agreement with experimental studies.     

Temperature and urea induced denaturation of the TRP‐cage mini protein TC5b: A simulation study consistent with experimental observations

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20‐residue Trp‐cage mini‐protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force‐field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR‐NOE atom–atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40–90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force‐field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side‐chain side‐chain contact (Trp6‐Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.     

β‐Hairpin folding and stability: molecular dynamics simulations of designed peptides in aqueous solution

The structural properties of a 10‐residue and a 15‐residue peptide in aqueous solution were investigated by molecular dynamics simulation. The two designed peptides, SYINSDGTWT and SESYINSDGTWTVTE, had been studied previously by NMR at 278 K and the resulting model structures were classified as 3:5 β‐hairpins with a type I + G1 β‐bulge turn. In simulations at 278 K, starting from the NMR model structure, the 3:5 β‐hairpin conformers proved to be stable over the time period evaluated (30 ns). Starting from an extended conformation, simulations of the decapeptide at 278 K, 323 K and 353 K were also performed to study folding. Over the relatively short time scales explored (30 ns at 278 K and 323 K, 56 ns at 353 K), folding to the 3:5 β‐hairpin could only be observed at 353 K. At this temperature, the collapse to β‐hairpin‐like conformations is very fast. The conformational space accessible to the peptide is entirely dominated by loop structures with different degrees of β‐hairpin character. The transitions between different types of ordered loops and β‐hairpins occur through two unstructured loop conformations stabilized by a single side‐chain interaction between Tyr2 and Trp9, which facilitates the changes of the hydrogen‐bond register. In agreement with previous experimental results, β‐hairpin formation is initially driven by the bending propensity of the turn segment. Nevertheless, the fine organization of the turn region appears to be a late event in the folding process. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic studies on the role of Lys-171 and Lys-358 in the beta subunit of sarcosine oxidase from Corynebacterium sp. U-96

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the beta subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent K(m) for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent K(m) for oxygen and K(d) of enzyme-sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.     

Exploring the differences and similarities between urea and thermally driven denaturation of bovine serum albumin: intermolecular forces and solvation preferences

The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea–BSA hydrogen bond energies were found to be weaker than water–BSA hydrogen bond energies. The greater strength of water–BSA hydrogen bonds than urea–BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea–protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.     

Characterization of the protein unfolding processes induced by urea and temperature

Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the β-sheets, while leaving the α-helices almost intact; on the contrary, high temperature preserved the β-sheets and led to a complete loss of the α-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replica-exchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent.     

Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP

We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180–190 K and is not affected by the proteins’ methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180–190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.     

Effect of urea on peptide conformation in water: molecular dynamics and experimental characterization

Molecular dynamics simulations of a ribonuclease A C-peptide analog and a sequence variant were performed in water at 277 and 300 K and in 8 M urea to clarify the molecular denaturation mechanism induced by urea and the early events in protein unfolding. Spectroscopic characterization of the peptides showed that the C-peptide analog had a high alpha-helical content, which was not the case for the variant. In the simulations, interdependent side-chain interactions were responsible for the high stability of the alpha-helical C-peptide analog in the different solvents. The other peptide displayed alpha-helical unwinding that propagated cooperatively toward the N-terminal. The conformations sampled by the peptides depended on their sequence and on the solvent. The ability of water molecules to form hydrogen bonds to the peptide as well as the hydrogen bond lifetimes increased in the presence of urea, whereas water mobility was reduced near the peptide. Urea accumulated in excess around the peptide, to which it formed long-lived hydrogen bonds. The unfolding mechanisms induced by thermal denaturation and by urea are of a different nature, with urea-aqueous solutions providing a better peptide solvation than pure water. Our results suggest that the effect of urea on the chemical denaturation process involves both the direct and indirect mechanisms.     

Dissecting the stability of a beta-hairpin peptide that folds in water: NMR and molecular dynamics analysis of the beta-turn and beta-strand contributions to folding.

NMR studies of the folding and conformational properties of a beta-hairpin peptide, several peptide fragments of the hairpin, and sequence-modified analogues, have enabled the various contributions to beta-hairpin stability in water to be dissected. Temperature and pH-induced unfolding studies indicate that the folding-unfolding equilibrium approximates to a two-state model. The hairpin is highly resistant to denaturation and is still significantly folded in 7 M urea at 298 K. Thermodynamic analysis shows the hairpin to fold in water with a significant change in heat capacity, however, DeltaCp degrees in 7 M urea is reduced. V/Y-->A mutations on one strand of the hairpin reduce folding to <10 %, consistent with a hydrophobic stabilisation model. We show that in a truncated peptide (residues 6-16) lacking the hydrophobic residues on one beta-strand, the type I' Asn-Gly turn in the sequence SINGKK is significantly populated in water in the absence of interstrand hydrophobic contacts. Unrestrained molecular dynamics simulations of unfolding, using an explicit solvation model, show that the conformation of the NG turn persists for longer than the AG analogue, which has a much lower propensity for type I' turn formation from a data base analysis of preferred turns. The origin of the high stability of the Asn-Gly turn is not entirely clear; data base analysis of 66 NG turns, together with molecular dynamics simulations, reveals no participation of the Asn side-chain in turn-stabilising interactions with the peptide backbone. However, hydration analysis of the molecular dynamics simulations reveals a pocket of "high density" water bridging between the Asn side-chain and peptide main-chain that suggests solvent-mediated interactions may play an important role in modulating phi,psi propensities in the NG turn region.     

Potentials of mean force for the interaction of blocked alanine dipeptide molecules in water and gas phase from MD simulations

We calculate potentials of mean force (PMFs) for the intermolecular interaction of two blocked alanine dipeptide (AcAlaNHMe) molecules in water and gas phase at two temperatures, 278 and 300 K, from all-atom molecular dynamics simulations. Simple models based on buried solvent accessible surface and one-dimensional potentials derived from distance-based radial distribution functions are not capable of expressing the short- and long-range complexity of the solute-solute interactions in water. Instead, radial and angular variations in the PMFs are observed with the two-dimensional potentials. The strength of the interactions for specific relative orientations of the molecules in the two-dimensional PMFs is more than double that observed in the one-dimensional PMFs. The populations of specific blocked alanine dipeptide conformations in water, such as alpha(R) and PPII, vary with temperature, and most significantly, with the distance between the centers of mass. A preference for helical conformations is observed at close encounter between molecules.     

The structural properties of magainin in water, TFE/water, and aqueous urea solutions: molecular dynamics simulations

Here, the MD simulations and comparative structural analysis of Magainin in water, TFE/water, and 2M, 4M, and BM urea solutions is reported. For MAG-TFE/water and MAG-2M urea the largely alpha helical conformation of the peptide is maintained throughout the 9-ns simulation. While in water, 4M urea, and 8M urea, the helix length decreases and at the same time helix radius increases. This suggests a more destabilized magainin secondary structure. Our simulation data reveals that the stabilizing effect of TFE is induced by preferential accumulation of TFE molecules around the alpha helical peptide. These results indicate that an aqueous urea solution solvates the surface of polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the peptide in comparison with water, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the peptide. At 8M urea, there are more direct interactions between the urea and solute, and the helix is destabilized. At 2M urea, the interaction of urea molecules and nonpolar residues are weak, therefore, the presence of urea molecules decreases the interactions of water molecules with hydrophilic groups. Urea could not deteriorate the peptide secondary structure with time from an initial helix structure.     

Temperature behaviour of viscous flow with proteins

The paper presents the results of viscosity determinations on aqueous solutions of different mammalian serum albumins at a wide range of concentrations and at temperatures ranging from 278 K to 318 K. On the basis of these measurements and a modified Arrhenius equation, the functional dependence of the solution activation energy of viscous flow on temperature was established. The analysis of the results obtained shows that the activation energy decreases with increasing temperature according to a square function for solutions, water molecules, and the albumins studied. The rate at which the activation energy decreases with increasing temperature is different for each albumin and mainly depends on its hydrodynamic radius.     

Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori

Helicobacter pylori's unique ability to colonize and survive in the acidic environment of the stomach is critically dependent on uptake of urea through the urea channel, HpUreI. Hence, HpUreI may represent a promising target for the development of specific drugs against this human pathogen. To obtain insight into the structure-function relationship of this channel, we developed conditions for the high-yield expression and purification of stable recombinant HpUreI. Detergent-solubilized HpUreI forms a homotrimer, as determined by chemical cross-linking. Urea dissociation kinetics of purified HpUreI were determined by means of the scintillation proximity assay, whereas urea efflux was measured in HpUreI-containing proteoliposomes using stopped-flow spectrometry to determine the kinetics and selectivity of the urea channel. The kinetic analyses revealed that urea conduction in HpUreI is pH-sensitive and saturable with a half-saturation concentration (or K(0.5)) of ~163 mM. The extent of binding of urea by HpUreI was increased at lower pH; however, the apparent affinity of urea binding (~150 mM) was not significantly pH-dependent. The solute selectivity analysis indicated that HpUreI is highly selective for urea and hydroxyurea. Removing either amino group of urea molecules diminishes their permeability through HpUreI. Similar to urea conduction, diffusion of water through HpUreI is pH-dependent with low water permeability at neutral pH.     

Effects of metal ion and solute conformation change on hydration of small amino acid

The effects of metal ion and solute conformation change on the structures, energetic and dynamics of water molecules in the first hydration shell of amino acid were studied, using three forms of alanine (Ala) and Li(+)/Ala as model molecules. The theoretical investigations were started with construction of the test-particle model (T-model) potentials for all molecules involved and followed by molecular dynamics (MD) simulations of [Ala](aq) and [Li(+)/Ala](aq) at 298 K. The MD results showed that the hydrogen bond (H-bond) networks of water at the functional groups of Ala are strengthened by the metal ion binding, whereas the rotation of the N-C(alpha) bond from the angle phi=0 degrees to 180 degrees brings about smaller effects which cannot be generalized. It was also shown that the dynamics of water molecule in the first hydration shell of amino acid could be estimated from the total-average potential energy landscapes and the water exchange diagrams. The MD results suggested inclusion of an additional dynamic step in the water exchange process, in which water molecule moves inside a channel within the first hydration shell of solute, before leaving the channel at some point. The theoretical results reported in the present work iterated the necessity to include explicit water molecules in the model calculations.     

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. This protein is structurally assembled as double dimers. Lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that wild-type protein had both dimers and monomers present in 2–4 M urea whilst only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folding through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs.     

A natural osmolyte trimethylamine N-oxide promotes assembly and bundling of the bacterial cell division protein, FtsZ and counteracts the denaturing effects of urea

Assembly of FtsZ was completely inhibited by low concentrations of urea and its unfolding occurred in two steps in the presence of urea, with the formation of an intermediate [Santra MK & Panda D (2003) J Biol Chem278, 21336-21343]. In this study, using the fluorescence of 1-anilininonaphthalene-8-sulfonic acid and far-UV circular dichroism spectroscopy, we found that a natural osmolyte, trimethylamine N-oxide (TMAO), counteracted the denaturing effects of urea and guanidium chloride on FtsZ. TMAO also protected assembly and bundling of FtsZ protofilaments from the denaturing effects of urea and guanidium chloride. Furthermore, the standard free energy changes for unfolding of FtsZ were estimated to be 22.5 and 28.4 kJ.mol(-1) in the absence and presence of 0.6 M TMAO, respectively. The data are consistent with the view that osmolytes counteract denaturant-induced unfolding of proteins by destabilizing the unfolded states. Interestingly, TMAO was also found to affect the assembly properties of native FtsZ. TMAO increased the light-scattering signal of the FtsZ assembly, increased sedimentable polymer mass, enhanced bundling of FtsZ protofilaments and reduced the GTPase activity of FtsZ. Similar to TMAO, monosodium glutamate, a physiological osmolyte in bacteria, which induces assembly and bundling of FtsZ filaments in vitro[Beuria TK, Krishnakumar SS, Sahar S, Singh N, Gupta K, Meshram M & Panda D (2003) J Biol Chem278, 3735-3741], was also found to counteract the deleterious effects of urea on FtsZ. The results together suggested that physiological osmolytes may regulate assembly and bundling of FtsZ in bacteria and that they may protect the functionality of FtsZ under environmental stress conditions.     

Urea perturbation and the reversibility of nucleohistone conformation

Urea effect on conformation and thermal stabilities in nucleohistone and NaCl-treated partially dehistonized nucleohistones has been studied by circular dichroism (CD) and thermal denaturation. Urea imposes a CD change at 278mm of DNA base pairs in native and NaCl-treated nucleohistones which can be decomposed into two parts: a decrease in Δε₂₇₈ for histone-free base pairs and an increase for histone-bound base pairs. The reduction by urea of Δε₂₂₀ of bound histones is approximately proportional to the increase of Δε₂₇₈ of histone-bound base pairs. Urea also lowers the melting temperatures of base pairs both free and bound by histones. The presence of urea indeed destroys the secondary structure of bound histones, causing changes in the conformation and thermal stabilities of histone-bound base pairs in nucleohistone. Such a urea perturbation on nucleohistone conformation is reversible.