Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.     

Long-term molecular dynamics simulation of copper azurin: structure, dynamics and functionality

A long-term molecular dynamics simulation (1.1 ns), at 300 K, of fully hydrated azurin has been performed to put into relationship the protein dynamics to functional properties with particular attention to those structural elements involved in the electron transfer process. A detailed analysis of the root mean square deviations and fluctuations and of the intraprotein H-bonding pattern has allowed us to demonstrate that a rigid arrangement of the beta-stranded protein skeleton is maintained during the simulation run, while a large mobility is registered in the solvent-exposed connecting regions (turns) and in the alpha-helix. Moreover, the structural elements, likely involved in the electron transfer path, show a stable H-bonding arrangement and low fluctuations. Analysis of the dynamical cross-correlation map has revealed the existence of correlated motions among residues connected by hydrogen bonds and of correlated and anti-correlated motions between regions which are supposed to be involved in the functional process, namely the hydrophobic patch and the regions close to the copper reaction center. The results are briefly discussed also in connection to the current through-bond tunneling model for the electron transfer process. Finally, a comparison with the structural and the dynamical behaviour of plastocyanin, whose structure and functional role are very similar to those of azurin, has been performed.     

Molecular dynamics simulation of solvated azurin: correlation between surface solvent accessibility and water residence times

A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent-accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67.     

Solvent effects on protein motion and protein effects on solvent motion. Dynamics of the active site region of lysozyme

The stochastic boundary molecular dynamics methodology is applied to the active site of the enzyme lysozyme. A comparison is made of in vacuo dynamics results from the stochastic boundary method and a full conventional molecular dynamics simulation of lysozyme. Excellent agreement between the two approaches is obtained. The influence of solvent on the residues in the active site region is explored and it is shown that both the structure and dynamics are affected. Of particular importance for the structure of the protein is the solvation of polar residues and the stabilization of like-charged ion pairs. The magnitude of the fluctuations is only slightly altered by the solvent; the overall increase in the root-mean-square fluctuations, relative to the vacuum run, is 11%. The solvent effect on dynamical properties is found not to be simply related to the solvent viscosity. Both the solvent exposure and dynamic aspects of protein-solvent interactions, including the relative time scales of the motions, are shown to play a role. The effects of the protein on solvent dynamics and structure are also observed to be significant. The solvent molecules around atoms in charged, polar and apolar side-chains show markedly different diffusion coefficients as well as exhibiting different solvation structures. One key example is the water around apolar groups, which is much less mobile than bulk water, or water solvating polar groups.     

Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by 1H-NMR chemical shift analysis

A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.     

Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.     

Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.     

The specificity in the interaction between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 is mainly determined by the copper protein

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.     

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.     

Solvent density and long-range dipole field around a DNA-binding protein studied by molecular dynamics

The distribution and orientation of solvent around a DNA-binding protein, 434 Cro, were investigated by molecular dynamics simulations with a periodic-boundary condition. The protein was treated in two states: charged and neutral. The computed high-density sites of the solvent around the protein correlated well with the experimentally determined crystal-water sites, in both the charged and neutral states. A local density map, introduced to investigate the solvent density around the highly mobile regions of the protein, showed a hydration shell around hydrophobic sidechains and hydrogen-bondable sites around hydrophilic sidechains, and also showed that the solvent density is sensitive to the slight concaves of the sidechain surface. The long-range solvent-dipole field was observed around the protein, where the pattern of the dipole ordering was considerably different between the charged and neutral states. A local solvent-dipole field was introduced, and the pattern of the dipole ordering was different between the hydrophobic and hydrophilic sidechains. The dipole field from the charged state provided a higher correlation to the electrostatic field obtained from the Poisson-Boltzmann's equation than that from the neutral state, although the correlation become weak quickly for the both states with increasing the protein-solvent distance.     

Electrostatic effects on electron-transfer kinetics in the cytochrome f-plastocyanin complex

In a complex of two electron-transfer proteins, their redox potentials can be shifted due to changes in the dielectric surroundings and the electrostatic potentials at each center caused by the charged residues of the partner. These effects are dependent on the geometry of the complex. Three different docking configurations (DCs) for intracomplex electron transfer between cytochrome f and plastocyanin were studied, defined by 1) close contact of the positively charged region of cytochrome f and the negatively charged regions of plastocyanin (DC1) and by (2, 3) close contact of the surface regions adjacent to the Fe and Cu redox centers (DC2 and DC3). The equilibrium energetics for electron transfer in DC1-DC3 are the same within approximately +/-0.1 kT. The lower reorganization energy for DC2 results in a slightly lower activation energy for this complex compared with DC1 and DC3. The long heme-copper distance (approximately 24 A) in the DC1 complex drastically decreases electronic coupling and makes this complex much less favorable for electron transfer than DC2 or DC3. DC1-like complexes can only serve as docking intermediates in the pathway toward formation of an electron-transfer-competent complex. Elimination of the four positive charges arising from the lysine residues in the positive patch of cytochrome f, as accomplished by mutagenesis, exerts a negligible effect (approximately 3 mV) on the redox potential difference between cyt f and PC.     

A molecular dynamics simulation of SNase and its hydration shell at high temperature and high pressure

Temperature- and pressure-induced unfolding of staphylococcal nuclease (SNase) was studied by Royer, Winter et al. using a variety of experimental techniques (SAXS, FT-IR and fluorescence spectroscopy, DSC, PPC, densimetry). For a more detailed understanding of the underlying mechanistic processes of the different unfolding scenarios, we have carried out a series of molecular dynamics (MD) computer simulations on SNase. We investigated the initial changes of the structure of the protein upon application of pressure (up to 5 kbar) and discuss volumetric and structural differences between the native and pressure pre-denatured state. Additionally, we have obtained the compressibility of the protein and hydration water and compare these data with experimental results. As water plays a crucial role in determining the structure, dynamics and function of proteins, we undertook a detailed analysis of the structure of the interfacial water and the protein-solvent H-bond network as well. Moreover, we report here also MD results on the temperature-induced unfolding of SNase. The time evolution of the protein volume and solvent accessible surface area during thermal unfolding have been investigated, and we present a detailed discussion of the temperature-induced unfolding pathway of SNase in terms of secondary and tertiary structural changes.     

Cytochrome P450 52A3: Modelling of 3D Structure and Surface Mutations

Abstract

Earlier W.-H. Schunck et al. [1] have prepared a water soluble enzymatically active fragment of cytochrome P450 52A3 (CYP52A3) which is lack of 66 amino acid residues, existed as a dimer in aqueous solution. Now we propose 3D structure of the fragment, which is based on multiple sequence alignment of the CYP52A3 with its homologues proteins of known 3D structure: CYP101, 102, 107A1 and 108. The structural model have been optimised and used as a prototype for computer simulation of point mutations. These mutations should bring some changes in the surface properties, interfering dimer formation. For this aim the point of 22 hydrophobic amino acid residues have been sequentially replaced with that of charged amino acids (GLU, ASP, ARG and LYS). The scoring of “mutants” was conducted based on the changes of protein surface hydrophobicity and protein-solvent interaction energy. An analysis of the surface hydrophobicity and protein-solvent interactions permit to select most sensitive three sites (171, 352 and particularly 164 amino acid residues). The dimerization of the following “mutant” fragments must be investigated experimentally.     

1H-NMR sequential assignments and cation-binding studies of spinach plastocyanin

The essentially complete assignment of the 1H-NMR spectrum of the Cu(i) form of spinach plastocyanin has been achieved using two-dimensional NMR techniques and sequence-specific resonance assignment procedures. A variety of pH and temperature conditions was utilised to overcome the problems of resonance overlap in the spectrum, degeneracy of C alpha H and solvent H2O chemical shifts, and cross-saturation of labile NH resonances. A qualitative analysis of the long-range nuclear Overhauser effects observed indicates that the backbone fold of spinach plastocyanin is very similar to that of poplar plastocyanin, whose structure has been solved by X-ray crystallography and differs in 22 of its 99 amino acid residues. The assignments provide a basis for further investigations into the structural and ion- and protein-binding properties of plastocyanin in solution.     

T4溶菌酶晶体分子堆积的研究

以不对称单位中只有一个分子的１０种不同晶型的Ｔ４溶菌酶晶体为材料,对晶体中的分子堆积进行了研究,结果表明,在溶剂含量较高的晶型中,非极性基团在接触面积中所占的比例略高于溶剂含量较低的晶型,而其极性和带电荷基团在接触面积中所占的比例略低于溶剂含量较低的晶型。溶剂含量较高的晶型多含有晶体学二重轴,二重轴相关的分子间的接触与其他接触相比,含有较少的极性相互作用。这些结果说明溶剂含量的高低可能是由不同结晶     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700+ reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6-8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42-45 and Nos. 59-61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42-45 and Nos. 59-61. Modification of plastocyanin at residues Nos. 42-45 showed no effect on the rate of P-700+ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700+ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59-61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 +/- 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42-45 or Nos. 59-61.     

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.     

Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5-bisphosphate

Gelsolin is an actin-severing protein whose action is initiated by Ca(2+) and inhibited by binding to phosphorylated inositol lipid or phosphoinositides. The regions of gelsolin responsible for phosphoinositide binding are comprised of residues 150-169 (G150-169) and 135-142 (G135-142). The corresponding peptides possess similar binding potency as native gelsolin. Their common feature is the presence of arginine and lysine residues that can bind to negatively charged phosphate groups of phosphoinositides. In this work the binding of the G150-169 peptide to a phosphatidylinositol 4,5-bisphosphate (PIP2) cluster in a lipid membrane model was investigated by molecular dynamics calculations (MD) with the AMBER 4.1 force field, taking into account explicit solvent molecules. Initially the structure of G150-169 was simulated by using the electrostatically driven Monte Carlo (EDMC) and MD methods, and the resulting structure agreed within 3.7 A backbone-atom root mean square deviation with the corresponding experimentally derived structure (PDB code: 1SOL). Using this model for the peptide, a subsequent MD simulation of G150-169 in a periodic box containing a model of dimyristoyl-phosphatidylcholine (DMPC) lipids with a cluster of four PIP2 molecules was carried out. During the simulation G150-169 interacted strongly with PIP2 molecules, initially by formation of salt bridges between its N-terminal basic groups and the phosphate groups of PIP2, followed by formation of hydrophobic bonds between the hydrophobic side chains of the peptide and the fatty acid tail of the lipid. As a result of the formation of hydrophobic bonds, the PIP2 molecules were pulled out from the lipid bilayer. This mode of binding differs from those of other PIP2-binding protein motifs such as PH domains that interact solely with the hydrophilic head group of PIP2. These results suggest that dissociation of gelsolin from actin by PIP2 lipids may involve entering of the PIP2 molecules to the gelsolin-actin interface, thereby weakening the interactions between these proteins.