Parameterization of peptide 13C carbonyl chemical shielding anisotropy in molecular dynamics simulations.

NMR chemical shielding anisotropy (CSA) relaxation is an important tool in the study of dynamical processes in proteins and nucleic acids in solution. Herein, we investigate how dynamical variations in local geometry affect the chemical shielding anisotropy relaxation of the carbonyl carbon nucleus, using the following protocol: 1) Using density functional theory, the carbonyl (13)C' CSA is computed for 103 conformations of the model peptide group N-methylacetamide (NMA). 2) The variations in computed (13)C' CSA parameters are fitted against quadratic hypersurfaces containing cross terms between the variables. 3) The predictive quality of the CSA hypersurfaces is validated by comparing the predicted and de novo calculated (13)C' CSAs for 20 molecular dynamics snapshots. 4) The CSA fluctuations and their autocorrelation and cross correlation functions due to bond-length and bond-angle distortions are predicted for a chemistry Harvard molecular mechanics (CHARMM) molecular dynamics trajectory of Ca(2+)-saturated calmodulin and GB3 from the hypersurfaces, as well as for a molecular dynamics (MD) simulation of an NMA trimer using a quantum mechanically correct forcefield. We find that the fluctuations can be represented by a 0.93 scaling factor of the CSA tensor for both R(1) and R(2) relaxations for residues in helix, coil, and sheet alike. This result is important, as it establishes that (13)C' relaxation is a valid tool for measurement of interesting dynamical events in proteins.     

Structural properties of an artificial protein that regulates the nucleation of inorganic and organic crystals

Technological advances have facilitated the generation of artificial proteins that possess the capabilities of recognizing and binding to inorganic solids and/or controlling nucleation processes that form inorganic solids. However, very little is known regarding the structure of these interesting polypeptides and how their structure contributes to functionality. To address this deficiency, we report structural investigations of an artificial protein, p288, that self-assembles and controls the nucleation of simple salts and organic compounds into dendrite-like crystals. Under aqueous conditions at low pH and in the presence of high salt, p288 is conformationally labile and exists primarily as a random coil conformer in equilibrium with other undefined secondary structures, including polyproline type II and beta turn. We note that p288 can fold into either a partial beta strand (at neutral pH) or a predominantly alpha helical (in the presence of TFE) conformation. Solid-state 13C-15N NMR experiments also reveal that p288 in the lyophilized, hydrated state possesses some degree of nonrandom coil structure. These results indicate that p288 is conformationally labile but can undergo conformational transitions to a more stable structure when water solvent loss/displacement occurs and protein concentrations increase. We believe that conformational instability and the ability to adopt different structures as a function of different environmental conditions represent important molecular features that impact p288 supramolecular assembly and crystal nucleation processes.     

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.     

Preparation and decarboxylative rearrangement of (Z)-enyne esters

A method to assemble (Z)-enyne esters via palladium-catalyzed cross coupling reactions of enol tosylates is reported. A base-mediated one-pot decarboxylative rearrangement of the enynes to enones is described. The scope of this process is examined.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.     

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Syntheses and conformational analyses of some 3-amino-2,5-dioxo-2,3,4,5-tetrahydro-1H-1-benzazepine derivatives: X-ray crystal structure of 35–3-[[(1,1-dimethylethoxy)carbonyl]amino]-2,5-dioxo-2,3,4,5-tetrahydro-1H-1-benzazepine

The constrained dipeptide mimic 1 was synthesized from 2 in three steps with 65% overall yield. Analyses of the ¹H nmr data of a number of 3-amino-2,5-dioxo-2,3,4,5-tetrahydro-1H-1-benzazepine derivatives led to the conclusion that these compounds adopt a similar conformation and that this ring system is rigid. X-ray crystallography was used to define the structure of 3 , and computer-aided energy minimization of 6 gave a preferred conformation similar to that observed in the crystal of 3 .