Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative‐scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active‐site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3), a highly active His‐ligated variant of P450‐BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3‐Hstar, catalyzes the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non‐natural enzyme activity.     

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.     

Sum-frequency generation: polarization surface spectroscopy analysis of the vibrational surface modes on the basal face of ice I(h)

In recent years, sum-frequency generation (SFG) has been used to investigate numerous interfaces including aqueous interfaces. A longstanding challenge to interpretation of the SFG results, along with the related aqueous-solution infrared and Raman spectra, is a lack of connection between features in the broad hydrogen-bonded region and molecular-level interactions or configurations. This paper reports results of a newly developed polarization analysis of the generated sum-frequency signal as a function of wavelength both to deconvolute spectral resonances and to characterize the dynamic polarization associated with the resonances. Operationally, the polarization angle of the generated sum frequency is determined by identifying the null angle. The technique is hence termed polarization-angle null analysis or PAN. PAN applied to ice is very powerful; it reveals that the hydrogen-bonded region of the basal face of ice I(h) contains at least five oscillators, each with a distinct polarization. The dynamic polarizability of the longest wavelength oscillator is nearly entirely transverse (perpendicular to the surface normal, i.e., in the surface plane); in contrast, the shortest wavelength oscillator is almost entirely longitudinal (along the surface normal).     

Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 x 10(-4)cm(2) V(-1) s(-1). The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.     

Cases,clusters, densities: Modeling the nonlinear dynamics of complex health trajectories

In the health informatics era, modeling longitudinal data remains problematic. The issue is method: health data are highly nonlinear and dynamic, multilevel and multidimensional, comprised of multiple major/minor trends, and causally complex—making curve fitting, modeling, and prediction difficult. The current study is fourth in a series exploring a case‐based density (CBD) approach for modeling complex trajectories, which has the following advantages: it can (1) convert databases into sets of cases (k dimensional row vectors; i.e., rows containing k elements); (2) compute the trajectory (velocity vector) for each case based on (3) a set of bio‐social variables called traces; (4) construct a theoretical map to explain these traces; (5) use vector quantization (i.e., k‐means, topographical neural nets) to longitudinally cluster case trajectories into major/minor trends; (6) employ genetic algorithms and ordinary differential equations to create a microscopic (vector field) model (the inverse problem) of these trajectories; (7) look for complex steady‐state behaviors (e.g., spiraling sources, etc) in the microscopic model; (8) draw from thermodynamics, synergetics and transport theory to translate the vector field (microscopic model) into the linear movement of macroscopic densities; (9) use the macroscopic model to simulate known and novel case‐based scenarios (the forward problem); and (10) construct multiple accounts of the data by linking the theoretical map and k dimensional profile with the macroscopic, microscopic and cluster models. Given the utility of this approach, our purpose here is to organize our method (as applied to recent research) so it can be employed by others. © 2015 Wiley Periodicals, Inc. Complexity 21: 160–180, 2016     

Porphyrin-based photocatalytic lithography

Photocatalytic lithography couples light with photoreactive coated mask materials to pattern surface chemistry. We excite porphyrins to create radical species that photocatalytically oxidize, and thereby pattern, chemistries in the local vicinity. The technique advantageously is suited for use with a wide variety of substrates. It is fast and robust, and the wavelength of light does not limit the resolution of patterned features. We have patterned proteins and cells to demonstrate the utility of photocatalytic lithography in life science applications.     

Faunal migration in late-glacial central Italy: implications for human resource exploitation

The hunter-gatherer transhumance model presents foragers as specialised hunters of migratory ungulates, which moved seasonally between coastal lowlands and interior uplands. We studied six animal teeth of horse (Equus hydruntinus) and red deer (Cervus elaphus) from four different archaeological sites: the Grotta di Vado all'Arancio, Grotta di Settecannelle, Grotta Polesini and Grotta di Pozzo, in central Italy to test whether the migratory patterns and seasonal variations recorded in their teeth were consistent with expectations of the transhumance model for this region during the late Upper Palaeolithic. Sequential sub-samples of enamel were analysed from each tooth for oxygen, carbon and strontium isotope ratios to reconstruct mobility and yearly seasonal variations. The results show little evidence that these animals were moving over different geological terrains throughout the year, although small variations in Sr isotope ratios and concentrations were detected that corresponded to probable seasonal variations as shown by variability in oxygen isotope sequences. The results do, however, demonstrate that Cervus elaphus and Equus hydruntinus had different ranging behaviours, with the former moving over wider areas than the latter. This methodology produces results appropriate to assess animal migratory behaviour and, in turn, to test the consistency of proposed models of hunter-gatherer subsistence and mobility strategies.     

Coupling of complex aromatic ring vibrations to solvent through hydrogen bonds: effect of varied on-ring and off-ring hydrogen-bonding substitutions

In this study, we examine the coupling of a complex ring vibration to solvent through hydrogen-bonding interactions. We compare phenylalanine, tyrosine, l-dopa, dopamine, norepinephrine, epinephrine, and hydroxyl-dl-dopa, a group of physiologically important small molecules that vary by single differences in H-bonding substitution. By examination of the temperature dependence of infrared absorptions of these molecules, we show that complex, many-atom vibrations can be coupled to solvent through hydrogen bonds and that the extent of that coupling is dependent on the degree of both on- and off-ring H-bonding substitution. The coupling is seen as a temperature-dependent frequency shift in infrared spectra, but the determination of the physical origin of that shift is based on additional data from temperature-dependent optical experiments and ab initio calculations. The optical experiments show that these small molecules are most sensitive to their immediate H-bonding environment rather than to bulk solvent properties. Ab initio calculations demonstrate H-bond-mediated vibrational coupling for the system of interest and also show that the overall small molecule solvent dependence is determined by a complex interplay of specific interactions and bulk solvation characteristics. Our findings indicate that a full understanding of biomolecule vibrational properties must include consideration of explicit hydrogen-bonding interactions with the surrounding microenvironment.