Directed formation of micro- and nanoscale patterns of functional light-harvesting LH2 complexes

The precision placement of the desired protein components on a suitable substrate is an essential prelude to any hybrid "biochip" device, but a second and equally important condition must also be met: the retention of full biological activity. Here we demonstrate the selective binding of an optically active membrane protein, the light-harvesting LH2 complex from Rhodobacter sphaeroides, to patterned self-assembled monolayers at the micron scale and the fabrication of nanometer-scale patterns of these molecules using near-field photolithographic methods. In contrast to plasma proteins, which are reversibly adsorbed on many surfaces, the LH2 complex is readily patterned simply by spatial control of surface polarity. Near-field photolithography has yielded rows of light-harvesting complexes only 98 nm wide. Retention of the native optical properties of patterned LH2 molecules was demonstrated using in situ fluorescence emission spectroscopy.     

Response normalized liquid chromatography nanospray ionization mass spectrometry

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run. The data obtained from four different structural classes of compounds [vicriviroc (VCV), desloratadine (DL), tolbutamide, and cocaine] and their metabolites indicate that by maintaining the solvent composition unchanged across the HPLC run, the influence of the solvent environment on the ionization efficiency is minimized. In comparison to responses obtained from radiochromatograms, responses from conventional LC-ESI-MS overestimated the VCV and DL responses, respectively, by 6- and 20-fold. Although VCV and DL responses obtained using LC-NSI-MS are within 2- to 6-fold from the respective radiochromatographic responses, the response normalization modification results in nearly uniform LC-NSI-MS response for all compounds evaluated.     

Crosslinking and degradation of PVP hydrogels as a function of dose and PVP concentration

Swelling was performed on dried membranes, normal ones and dried gel to unravel the role of crosslinking and degradation on polymeric structure during drying and hydration process. The comparison of the swelling results suggested that the network were formed only by PVP molecules. The complex mixture of macromolecules showed a irreversible behavior upon drying and hydration, probably as a function of PEG and/or agar entangling in effective physical crosslink. The best network regularity and useful properties was found at 20–30 kGy.     

Application of combined Monte Carlo and molecular dynamics method to simulation of dipalmitoyl phosphatidylcholine lipid bilayer

We describe a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer. The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer, in a constant volume and temperature ensemble. The procedure is described in detail and is applied to a bilayer of 100 molecules of dipalmitoyl phosphatidylcholine (DPPC) and 3205 water molecules. We find that the hybrid simulation procedure enhances the equilibration of the bilayer as measured by the convergence of the area per molecule and the segmental order parameters, as compared with a simulation using only molecular dynamics (MD). Progress toward equilibration is almost three times as fast in CPU time, compared with a purely MD simulation. Equilibration is complete, as judged by the lack of energy drift in three separate 200-ps runs of continuous MD started from different initial states. Results of the simulation are presented and compared with experimental data and with other recent simulations of DPPC. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1153–1164, 1999     

Investigation of calcium-induced, noncovalent association of calmodulin with melittin by electrospray ionization mass spectrometry

Noncovalent association of Ca²⁺-loaded calmodulin with a target peptide melittin was studied by electrospray ionization mass spectrometry (ESI-MS). ESI-MS does not reveal any binding of the apocalmodulin to the melittin. Partial loading of calmodulin with calcium leads to weak association with melittin. Upon binding of two calcium ions to the protein, changes in the conformation of calmodulin occur; these changes are sufficient to promote binding of melittin. Saturation of the protein with Ca²⁺ (a distribution of up to seven calcium ions is detected) induces a large increase of the binding to melittin. The stoichiometry of peptide binding to calmodulin is 1:1.     

Single-molecule spectroscopy of fluorescent proteins

The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the single-molecule level, which removes averaging inherent to ensemble studies. In this paper we review how single-molecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.

Controlling the Structure of Manganese(II) Phosphates by the Choice and Ratio of Organophosphate and Auxiliary Ligands

Tetranuclear manganese(II) phosphates [Mn(dipp)(bpy)]₄?4 H₂O ( 1 ) and [Mn₄(dmpp)₂(dmppH)₄(bpy)₄(H₂O)₂]?H₂O ( 2 ) have been prepared from Mn(OAc)₂?4 H₂O and 2,6‐diisopropylphenyl phosphate (dippH₂) or 2,6‐dimethylphenyl phosphate (dmppH₂) in the presence of 2,2′‐bipyridine (bpy). In contrast, the reaction between [Mn(bpy)₂(OAc)(ClO₄)]?H₂O and dippH₂ affords [Mn(bpy)₂(dippH)]₂?2 ClO₄?2 CH₃OH ( 3 ). The reactions of Mn(OAc)₂?4 H₂O, dippH₂, and pyridine (py) or 3,5‐dimethylpyrazole (dmpz) in CH₃CN under reflux afford hexanuclear complexes [Mn₆(dipp)₆(py)₈]?2CH₃CN ( 4 ) and [Mn₆(dipp)₆(dmpz)₆(AcOH)₂]?2 H₂O ( 5 ), respectively. Although compounds 1 and 2 are tetrameric, the former is a closed cubane‐like structure resembling the D4R secondary building unit of zeolites, whereas the latter exists in a staircase structure with fused Mn₂O₄P₂ rings. The core structure of 3 contains a Mn₂O₄P₂ eight‐membered ring that resembles the S4R building block of zeolites. Single‐crystal X‐ray diffraction studies reveal that compounds 4 and 5 have a similar core structure and differ from each other by the neutral ligands coordinated to manganese ions. All six phosphate ligands exist in a doubly deprotonated [(RO)PO₃^2?] form and exhibit two types of binding modes [5.222] and [3.111]. An interesting feature of compounds 1 – 5 is that although they are oligonuclear complexes, there is an absence of oxido bridges. The magnetic properties of compounds 1 – 5 have been investigated in the temperature range 5–298 K, and it was found that all the compounds obey the Curie law.     

Adsorption and photocatalytic degradation of methylene blue using high surface area titanate nanotubes (TNT) synthesized via hydrothermal method

Removal of methylene blue (MB) via adsorption and photocatalysis using titanate nanotubes (TNTs) with different surface areas were investigated and compared to commercial titanium dioxide (TiO₂) P25 Degussa nanoparticles. The TNTs with surface area ranging from 20 m²/g to 200 m²/g were synthesized via hydrothermal method with different reaction times. TEM imaging confirmed the tubular structure of TNT while XRD spectra indicated all TNTs exhibited anatase crystallinity. Batch adsorption rate showed linearity with surface properties of TNTs, where materials with higher surface area showed higher adsorption rate. The highest MB adsorption (70%) was achieved by TNT24 in 60 min whereas commercial TiO₂ exhibited the lowest adsorption of only 10% after 240 min. Adsorption isotherm studies indicated that adsorption using TNT is better fitted into Langmuir adsorption isotherm than Freundlich isotherm model. Furthermore, TNT24 was able to perform up to 90% removal of MB within 120 min, demonstrating performance that is 2-fold better compared to commercial TiO₂. The high surface area and surface Bronsted acidity are the main reasons for the improvement in MB removal performance exhibited by TNT24. The improvement in surface acidity enhanced the adsorption properties of all the nanotubes prepared in this study.     

A regioselective multicomponent protocol for the synthesis of novel bioactive 4-hydroxyquinolin-2(1H)-one grafted monospiropyrrolidine and thiapyrrolizidine hybrids

An expedient route toward the synthesis of 4-hydroxyquinolone grafted spiropyrrolidines or pyrrolizidines has been accomplished through 1,3-dipolar cycloaddition reaction of various azomethine ylides derived from isatin or acenaphthalene and sarcosine with 4-hydroxyquinolone derivatives as dipolarophile. The regio and stereo chemical outcome of the cycloaddition reaction is ascertained by X-ray crystallographic studies and spectroscopic techniques of the cycloadducts. Furthermore, cytotoxicity evaluation of selected compounds showed significant inhibition of cell proliferation against cervical as well as colon cancer cell lines.