烟草薄层培养生长素结合蛋白ABP1的定位和ABP1在细胞分化中的变化

以烟草（Nicotiana tabacum L.)盛花期花梗薄层为材料,研究营养芽分化的不同时期生长素结合蛋白（ABP1)在组织与细胞中的分布变化,免疫荧光标记结果表明,烟草花梗中ABP1主要分布于表皮及亚表皮1－2层细胞内。不同分化期ABP1在烟草花梗薄层原生质体中的表达不同,细胞分化旺盛期ABP1的表达最强,分化后期ABP1的表达有所减弱;Western blotting结果表明,ABP1多克隆抗血清与烟草花梗薄层细胞及分化过程中26kD蛋白有免疫交叉反应。     

Distribution and Expression of Auxin Binding Protein 1 (ABP1) in the Thin Cell Layers of Tobacco

Tobacco （ Nicotiana tabacum L.） pedicel in the blooming period was used to study the distribution of auxin binding protein 1 (ABP1) in the tobacco tissue and cell at different time in the culture of thin cell layers (TCL). Using the immunofluorescence marker it was indicated that the main ABP1 was distributed in the epidermis and the 1st and 2nd layers of the subepidermis cells. A little ABP1 was distributed in the cortex. Tobacco ABP1 was induced to express in the protoplast of tobacco pedicel TCL cultured in MS culture medium containing IAA and BA. Expression of ABP1 in the protoplast was stronger in the active period of vegetative bud differentiation. ABP1 expression became weaker in later period of differentiation. The result of SDS-PAGE and Western blotting showed the molecular weight of tobacco ABP1 in TCL was 26 kD.     

Cholesterol-sphingomyelin interactions: a molecular dynamics simulation study

Stearoylsphingomyelin (SSM) bilayers containing 0, 22, and 50 mol % cholesterol (Chol) and a pentadecanoyl-stearoylphosphatidylcholine (15SPC) bilayer containing 22 mol % Chol were molecular dynamics simulated at two temperatures (37 degrees C and 60 degrees C). 15SPC is the best PC equivalent of SSM. The Chol effect on the SSM bilayer differs significantly from that on the 15SPC bilayer. At the same temperature and Chol content, H-bonding of Chol with SSM is more extensive than with 15SPC. SSM-Chol H-bonding anchors the OH group of Chol in the lower regions of the SSM-Chol bilayer interface. Such a location strengthens the influence of Chol on the SSM chains. In effect, the phase of the SSM-Chol bilayer containing 22 mol % Chol at 37 degrees C is shifted from the gel to the liquid-ordered phase, and the bilayer displays similar properties below and above the main phase-transition temperature for a pure SSM bilayer of approximately 45 degrees C. In contrast, due to a higher location, Chol is not able to change the phase of the 15SPC-Chol bilayer, which at 37 degrees C remains in the gel phase. Chol affects both the core and interface of the SSM bilayer. With increasing Chol content, the order of SSM chains and hydration of SSM headgroups increase, whereas polar interactions between lipids decrease.     

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

拟南芥生长素结合蛋白ABP1 的原核表达及蛋白纯化

目的:鉴于生长素结合蛋白(Auxin Binding Protein,ABP)能与生长素特异性结合,因而探讨研究其直接用于生长素信号转导机理和生物传感器的可能性与可行性。方法:通过RT-PCR获得拟南芥生长素结合蛋白1(Auxin bing protein 1,ABP1)的全长CDS,将其克隆到原核表达载体pGEX4T-1中,成功构建pGEX4T-1-ABP1重组表达载体。经酶切、PCR及DNA测序鉴定后,将阳性质粒转化表达受体菌BL21(DE3)。加入异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导后,取样进行SDS-PAGE分析。结果:成功表达出一个分子量约为43 kD的可溶性融合蛋白,并利用GST亲和柱纯化方式得到了ABPl。结论:通过原核表达并经GST柱纯化后获得ABP1,为生长素生物传感器的研制开辟新的途径。同时为进一步研究ABP1与生长素的信号转导机制和生长素在生物传感测定技术中的研究和应用奠定基础。     

The structure and dynamics of ACTH (1-10) on the surface of a sodium dodecylsulfate (SDS) micelle: a molecular dynamics simulation study

ACTH (1-10), an adrenocorticotropin hormone fragment, was studied by molecular dynamics (MD) simulation in the NPT ensemble in an explicit sodium dodecylsulfate (SDS) micelle. Initially, distance restraints derived from NMR nuclear Overhauser enhancements were incorporated during the equilibration stage of the simulation. The analyses of the trajectories from the subsequent unrestrained MD showed that ACTH (1-10) does not conform to a helical structure at the micelle-water interface; however, the structure is amphipathic. The loss of the helical structure is due to decreased intramolecular hydrogen bonding accompanied by an increase of hydrogen bonding between the amide hydrogens of the peptide and the micelle head-groups. ACTH (1-10) was found to lie on the surface of the SDS micelle. Most of the hydrophobic interactions came from the side-chains of Met-4, Phe-7 and Trp-9. The peptide bonds were either hydrated or involved in intramolecular hydrogen bonding. Decreased hydration for the backbone of His-6 and Phe-7 was due to intermolecular hydrogen bonding with the SDS head-groups. The time correlation functions of the N-H bonds of the peptide in water and in the micelle showed that the motions of the peptide, except for the N- and C-termini, are significantly reduced when partitioned in the micelle.     

A molecular dynamics study of a miRNA:mRNA interaction

In this paper we present a methodology to evaluate the binding free energy of a miRNA:mRNA complex through molecular dynamics (MD)–thermodynamic integration (TI) simulations. We applied our method to the Caenorhabditis elegans let-7 miRNA:lin-41 mRNA complex—a validated miRNA:mRNA interaction—in order to estimate the energetic stability of the structure. To make the miRNA:mRNA simulation possible and realistic, the methodology introduces specific solutions to overcome some of the general challenges of nucleic acid simulations and binding free energy computations that have been discussed widely in many previous research reports. The main features of the proposed methodology are: (1) positioning of the restraints imposed on the simulations in order to guarantee complex stability; (2) optimal sampling of the phase space to achieve satisfactory accuracy in the binding energy value; (3) determination of a suitable trade-off between computational costs and accuracy of binding free energy computation by the assessment of the scalability characteristics of the parallel simulations required for the TI. The experiments carried out demonstrate that MD simulations are a viable strategy for the study of miRNA binding characteristics, opening the way to the development of new computational target prediction methods based on three-dimensional structure information.     

Pressure denaturation of apomyoglobin: a molecular dynamics simulation study

The effect of pressure on the structure and mobility of Sperm Wale Apomyoglobin was studied by Molecular Dynamics computer simulation at 1 bar and 3 kbar (1 atm=1.01325 bar=101.325 kPa). The results are in good agreement with the available experimental data, allowing further analysis of other features of the effect of pressure on the protein solution. From the analysis of Secondary Structures (SS) along the trajectories it is observed that alpha-helixes are favoured under pressure at the expense of bends, turns and 3-helixes. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. The studies of tertiary structure show important conformational changes. The evolution of the Solvent Accessed Surface (SAS) with pressure shows a notorious increase due almost completely to a biased raise in the hydrophobic area exposed, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.     

Combination of anti-hypertensive drugs: a molecular dynamics simulation study

The anti-hypertensive drugs amlodipine, atenolol and lisinopril, in ordinary and PEGylated forms, with different combined-ratios, were studied by molecular dynamics simulations using GROMACS software. Twenty simulation systems were designed to evaluate the interactions of drug mixtures with a dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane, in the presence of water molecules. In the course of simulations, various properties of the systems were investigated, including drug location, diffusion and mass distribution in the membrane; drug orientation; the lipid chain disorder as a result of drug penetration into the DMPC membrane; the number of hydrogen bonds; and drug surface area. According to the results obtained, combined drugs penetrate deeper into the DMPC lipid bilayer membrane, and the lipid chains remain ordered. Also, the combined PEGylated drugs, at a combination ratio of 1:1:1, enhance drug penetration into the DMPC membrane, reduce drug agglomeration, orient the drug in a proper angle for easy penetration into the membrane, and decrease undesirable lipotoxicity due to distorted membrane self-assembly and thickness.

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Photoinduced conformational dynamics of a photoswitchable peptide: a nonequilibrium molecular dynamics simulation study

Employing nonequilibrium molecular dynamics simulations, a comprehensive computational study of the photoinduced conformational dynamics of a photoswitchable bicyclic azobenzene octapeptide is presented. The calculation of time-dependent probability distributions along various global and local reaction coordinates reveals that the conformational rearrangement of the peptide is rather complex and occurs on at least four timescales: 1) After photoexcitation, the azobenzene unit of the molecule undergoes nonadiabatic photoisomerization within 0.2 ps. 2) On the picosecond timescale, the cooling (13 ps) and the stretching (14 ps) of the photoexcited peptide is observed. 3) Most reaction coordinates exhibit a 50-100 ps component reflecting a fast conformational rearrangement. 4) The 500-1000 ps component observed in the simulation accounts for the slow diffusion-controlled conformational equilibration of the system. The simulation of the photoinduced molecular processes is in remarkable agreement with time-resolved optical and infrared experiments, although the calculated cooling as well as the initial conformational rearrangements of the peptide appear to be somewhat too slow. Based on an ab initio parameterized vibrational Hamiltonian, the time-dependent amide I frequency shift is calculated. Both intramolecular and solvent-induced contributions to the frequency shift were found to change by < or = 2 cm(-1), in reasonable agreement with experiment. The potential of transient infrared spectra to characterize the conformational dynamics of peptides is discussed in some detail.     

Expression and Subcellular Localization of Arabidopsis thaliana Auxin-Binding Protein 1 (ABP1)

Expression of Arabidopsis thaliana ABP1 (AUXIN-BINDING PROTEIN 1) was studied using a promoter:GUS approach. Two promoter regions were analyzed. The 1585-bp promoter region upstream of the translation start site (P _ABP1 ) showed different activity compared to the promoter region that included, in addition, the first two introns and three exons of the transcribed ABP1 sequence (P _ABP1i1,2), indicating that cis elements were present downstream of the start codon. P _ABP1i1,2-driven β-glucuronidase activity was highest in growing leaves, in the root meristem, in vascular tissues, and in hydathodes. ABP1 promoter activities overlapped largely but not completely with that of DR5, which is a marker for the ARF-AuxRE-dependent auxin response. Subcellular ABP1 localization was studied using a constitutively overexpressed EGFP-ABP1 fusion protein. Results confirmed predominant localization to the endoplasmic reticulum as was concluded previously.     

水稻生长素结合蛋白ABP1的原核表达及纯化

生长素结合蛋白能够与生长素特异性结合,因而有可能直接被用作生长素免疫分析和生物传感测定中的高特异性、高亲和力识别分子.本研究通过RT-PCR获得水稻生长素结合蛋白1(ABP1)cDNA,将其克隆到原核表达载体pET-32a(+)中,成功构建pET-32a-ABP1 重组表达载体.经酶切、PCR及DNA测序鉴定后,将阳性...     

Concentration effect of cimetidine with POPC bilayer: a molecular dynamics simulation study

All-atom molecular dynamics simulations have been performed on cimetidine in the presence of a palmitoyloleoylphosphatidylcholine (POPC) bilayer. The free energy profile of a single cimetidine molecule passing across POPC bilayer displays a minimum at the interface of bilayer and water. Ten cimetidine molecules were inserted into POPC bilayer to obtain an 8 mol % drug model, and molecular dynamics results showed that cimetidine molecules reside at the polar region of POPC bilayer with sulphur atoms directing to the hydrophobic region. By comparing the one drug model with 8 mol % drug model, one can see that the central barrier to cross the membrane increases while the free energy in bulk water decreases, indicating that the ability of cimetidine passing across the POPC bilayer weakens at increased concentration. In addition, the free energy minimum shifts closer to the hydrophobic core. Our results indicate that with the increased drug concentration, it is more difficult for cimetidine to enter and pass across POPC bilayer.     

Internal dynamics of d(CGCAAATTTGCG)2: a comparison of NMR relaxation measurements with a molecular dynamics simulation

We report the analysis of a 250 ps molecular dynamics simulation of the dodecamer d(CGCAAATTT-GCG)₂ immersed in a rectangular box of 3469 water molecules with 22 Na⁺ counterions. The internal dynamics of the molecule were investigated by studying the relevant autocorrelation functions related to the ¹³C-NMR relaxation parameters of the C1′-H1′ bonds of the sugar rings. The calculated effective correlation times τ _e (∼13 ps) and the order parameter S² (∼0.82) of the Lipari and Szabo formalism (Lipari and Szabo 1982a, b) are in satisfactory agreement with those determined previously by NMR (Gaudin et al. 1995, 1996). ¹H-¹H NOE buildups have also been measured experimentally and agree with those computed from the simulation. These results validate the simulation, and a more detailed analysis of the internal dynamics of the dodecamer was undertaken. Analysis of the distributions and of the autocorrelation functions of the glycosidic angle flucuations χ shows that the rotational motion of the sugar rings about their glycosidic bond conforms to a restricted diffusion mechanism. The amplitude of the motions and the diffusion constant are 20° and 17.10⁹ rad²s^–1 respectively. These values are in good agreement with ¹³C NMR data. Furthermore the simulation allows us to rule out another model also consistent with the experiment, consisting of a two-state jump between a syn and an anti conformation. Received: 19 November 1996 / Accepted: 17 March 1997     

Protein dynamics in solution and in a crystalline environment: a molecular dynamics study

The effect of a solvent and a crystalline environment on the dynamics of proteins is investigated by the method of computer simulation. Three 25-ps molecular dynamics simulations at 300 K of the bovine pancreatic trypsin inhibitor (BPTI), consisting of 454 heavy atoms, are compared: one of BPTI in vacuo, one of BPTI in a box with 2647 spherical nonpolar solvent atoms, and one of BPTI surrounded by fixed crystal image atoms. Both average and time-dependent molecular properties are examined to determine the effect of the environment on the behavior of the protein. The dynamics of BPTI in solution or in the crystal environment are found to be very similar to that found in the vacuum calculation. The primary difference in the average properties is that the equilibrium structure in the presence of solvent or the crystal field is significantly closer to the X-ray structure than is the vacuum result; concomitantly, the more realistic environment leads to a number density closer to experiment. The presence of solvent has a negligible effect on the overall magnitude of the positional or dihedral angle fluctuations in the interior of the protein; however, there are changes in the decay times of the fluctuations of interior atoms. For surface residues, both the magnitude and the time course of the motions are significantly altered by the solvent. There tends to be an increase in the displacements of long side chains and the flexible parts of the main chain that protrude into the solvent. Further, these motions tend to have a more diffusive character with longer relaxation times than in vacuo. The crystal environment has a specific effect on a number of side chains which are held in relatively fixed positions through hydrogen-bond and electric interactions with the neighboring protein atoms. Most of the effects of the solution environment seem to be sufficiently nonspecific that it may be possible to model them by applying a mean field and stochastic dynamic methods.     

Mechanism of beta-purothionin antimicrobial peptide inhibition by metal ions: molecular dynamics simulation study

Wheat beta-purothionin is a highly potent antimicrobial peptide which, however, is inactivated by metal ions. The key structural properties and mechanisms of inhibition of beta-purothionin were investigated for the first time using unconstrained molecular dynamics simulations in explicit water. A series of simulations were performed to determine effects of temperature and the metal ions. Analyses of the unconstrained simulations allowed the experimentally unavailable structural and dynamic details to be unambiguously examined. The global fold and the alpha1 helix of beta-purothionin are thermally stable and not affected by metal ions. In contrast, the alpha2 helix unfolds with shift of temperature from 300 K and in the presence of metal ions. The network of conserved residues including Arg30 and Lys5 is sensitive to environmental changes and triggers unfolding. Loop regions display high flexibility and elevated dynamics, but are affected by metal ions. Our study provides insights into the mechanism of metal ion-based inhibition.     

GD1a in phospholipid bilayer: a molecular dynamics simulation

Molecular dynamics simulation of ganglioside GD1a attached to the upper layer of a fully hydrated lipid bilayer of dimyristoyl phosphatidyl choline (DMPC) at room temperature under periodic boundary conditions was performed. The time average conformation of GD1a reveals that the terminal sialic acid is more exposed into the solvent than the internal branched one. Many interresidual contacts between N-acetyl galactosamine-internal branched sialic acid; external Gal-external sialic acid; N-acetyl galactosamine-internal gal are also observed. The conformation of the GD1-hexasaccharide is stabilized by a number of intra molecular hydrogen bonds that were previously observed experimentally. The simulation results indicate that the presence of a single GD1a molecule has local effects on the bilayer. A local disorder in the arrangement of the acyl chains as well as the head groups is evident in the upper layer due to the presence of GD1a.     

The dominant interaction between peptide and urea is electrostatic in nature: a molecular dynamics simulation study

The conformational equilibrium of a blocked valine peptide in water and aqueous urea solution is studied using molecular dynamics simulations. Pair correlation functions indicate enhanced concentration of urea near the peptide. Stronger hydrogen bonding of urea-peptide compared to water-peptide is observed with preference for helical conformation. The potential of mean force, computed using umbrella sampling, shows only small differences between urea and water solvation that are difficult to quantify. The changes in solvent structure around the peptide are explained by favorable electrostatic interactions (hydrogen bonds) of urea with the peptide backbone. There is no evidence for significant changes in hydrophobic interactions in the two conformations of the peptide in urea solution. Our simulations suggest that urea denatures proteins by preferentially forming hydrogen bonds to the peptide backbone, reducing the barrier for exposing protein residues to the solvent, and reaching the unfolded state.     

High-Temperature unfolding of a trp-Cage mini-protein: a molecular dynamics simulation study

Background  

Trp cage is a recently-constructed fast-folding miniprotein. It consists of a short helix, a 3,10 helix and a C-terminal poly-proline that packs against a Trp in the alpha helix. It is known to fold within 4 ns.