Gregg Whited

Genomics to fluxomics and physiomics — pathway engineering

Developments in microanalytical methods are enabling quantitative measurement of multiple metabolic fluxes and, in conjunction with transcript and proteomic profiling, are revolutionizing the ability of researchers to manipulate metabolism through pathway engineering in a variety of species. We review recent literature on the advances in genomics, proteomics, fluxomics and computational modeling focused on metabolic pathway engineering applications.

Whole-Cell Biotransformation of m -Ethyltoluene into 1 S ,6 R -5-Ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic Acid as an Approach to the C-Ring of the Binary IndoleIndoline Alkaloid Vinblastine.

The title compound 3, a potential building block for the construction of analogues of the clinically important anti-cancer agent vinblastine (1), has been prepared in an efficient manner through a whole-cell biotransformation of m-ethyltoluene (4) using the microorganism Pseudomonas putida BGXM1 which expresses the enzyme toluate dioxygenase (TADO). Metabolite 3 was readily converted into derivatives 5–8 using conventional chemical techniques and the structure, including absolute stereochemistry, of the last of these was established by single-crystal X-ray analysis.

Photochemical and Thermal Stability of Green and Blue Proteorhodopsins: Implications for Protein-Based Bioelectronic Devices

The photochemical and thermal stability of the detergent-solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR, respectively, are investigated to determine the viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential UV-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7 × 10(4) photocycles protein(-1), is 4-5 times more stable than BPR (9 × 10(3) photocycles protein(-1)), but is less stable than native bacteriorhodopsin (9 × 10(5) photocycles protein(-1)) or the 4-keto-bacteriorhodopsin analogue (1 × 10(5) photocycles protein(-1)). The thermal stabilities are assigned by using differential scanning calorimetry and thermal bleaching experiments. Both proteorhodopsins display excellent thermal stability, with melting temperatures above 85 °C, and remain photochemically stable up to 75 °C. The biological relevance of our results is also discussed. The lower cyclicity of BPR is found to be adequate for the long-term biological function of the host organism at ocean depths of 50 m or more.

Evaluation of blue and green absorbing proteorhodopsins as holographic materials.

Transient holographic diffraction is observed for the green (GPR) and blue (BPR) absorbing proteorhodopsins (BAC31A8 and HOT75M1, respectively), as well as the GPR E108Q and BPR E110Q variants. In contrast to bacteriorhodopsin, where the metastable bR-M pair is responsible for generating diffraction, the pR and red-shifted N-like states fulfill that role in both the green and blue wild-type proteorhodopsins. The GPR E108Q and BPR E110Q variants, however, behave more similarly to their bacteriorhodopsin analogue, D96N, with diffraction arising from the PR M-state (strongly enhanced in both GPR E108Q and BPR E110Q). Of the four proteins evaluated, wild type (WT) GPR and GPR E108Q produce the highest diffraction efficiencies, etamax, at approximately 1% for a 1.7 OD sample. GPR E108Q, however, requires 1-2 orders of magnitude less laser intensity to generate eta equivalent to WT GPR and BR D96N under similar conditions (as compared to literature values). WT BPR requires lower actinic powers than GPR but diffracts only about 30% as well. BPR E110Q performs the most poorly of the four, with etamax < 0.05% for a 1.4 OD film. The Kramers-Kronig transformation and Kogleniks coupled wave theory were used to predict the dispersion spectra and diffraction efficiency for the long M-state variants. To a first approximation, the gratings formed by all samples decay upon discontinuing the 520 nm actinic beams with a time constant characteristic of the appropriate intermediate: the N-like state for WT GPR and BPR and the M-state for GPR 108Q and BPR E110Q.

Chapter 7 Tools to Enhance Membrane Protein Crystallization

Publisher Summary This chapter describes a novel technique, self-interaction chromatography (SIC), to rapidly measure the second virial coefficient, B, (a thermodynamic term that provides semiquantitative information regarding protein–protein interactions) of proteins in a variety of cosolvents. SIC provides a rapid diagnostic for determining the optimum solution conditions that promote increased protein stability and minimize unwanted nonspecific aggregation. In addition, SIC allows the selection of specific solution conditions (including detergent type and concentration) for subsequent optimization of protein solubility, homogeneity, and crystallization. Balanced solubility and crystallization screens have been designed, such that B values are experimentally measured via SIC for a relatively small number of conditions, providing a balanced representation of the total number of possible combinations of chemical variables. The screen conditions plus corresponding B values are then used to train a predictive algorithm (neural net) that subsequently produces an in silico screen, predicting the B values for all possible combinations of the chemical variables. The high-positive B values indicate solution conditions that should provide increased protein solubility and consequently, minimization of nonspecific protein aggregation. Slightly negative B values indicate solution conditions that have a higher probability of yielding crystals. The combined use of these innovative technologies provides a powerful approach to address several of the critical impediments (i.e., protein production, stability, purification, and crystallization) preventing membrane structure determinations.