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Summary Using interferometry based biosensors the binding and release of eNOS and nNOS from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within about three orders of magnitude of the Smoluchowski limit imposed by orientation independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM binding site and the complementary surface on CaM. Protein kinase C (PKC) phosphorylation of eNOS T495, adjacent to the CaM binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca+2 binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca+2 dependence of CaM binding. Hence, changes in the Ca+2 sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca+2 sensitivity need not be tightly linked to CaM sensitivity of activation. This work will be extended using mutants to probe the roles of individual structural elements in binding and release.

bCaldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C 5 and C 6 sugars primarily to hydrogen gas, lactate, acetate, and CO 2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.T hermophilic bacteria of the genus Caldicellulosiruptor are currently under intense investigation due to their ability to decompose lignocellulosic plant biomass anaerobically at high temperature, thereby potentially mitigating costly thermochemical pretreatment steps (1, 2). One of these species, Caldicellulosiruptor bescii, has an optimal growth temperature of 78°C and is the most thermophilic cellulose degrader known to date. It is able to ferment high concentrations of cellulosic feedstock primarily to hydrogen gas, lactate, acetate, and CO 2 (3, 4). Species from this genus can degrade cellulose (and also xylan), using novel multidomain glycosyl hydrolases, representing a new paradigm in cellulose conversion by anaerobic thermophiles (2). Moreover, the recent development of a genetic system for C. bescii creates potential for using this and related species for consolidated biomass processing in the production of liquid fuels (5).Developing metabolic engineering strategies for any microorganism obviously requires an in-depth understanding of their primary metabolism. Evidence that C. bescii may have a completely uncharacterized aspect to its primary redox metabolism came from an analysis of its genome sequence for molybdoenzymes (6). These are present in virtually all forms of life, serving diverse roles in primary metabolism of carbon, nitrogen and sulfur (7). As expected, we found that the C. bescii genome contains genes necessary for the synthesis of the pyranopterin cofactor that coordinates molybdenum (Mo) in such enzymes (7). Accordin...

Caldicellulosiruptor bescii is an extremely thermophilic cellulolytic bacterium with great potential for consolidated bioprocessing of renewable plant biomass. Since it does not natively produce ethanol, metabolic engineering is required to create strains with this capability. Previous efforts involved the heterologous expression of the gene encoding a bifunctional alcohol dehydrogenase, AdhE, which uses NADH as the electron donor to reduce acetyl-CoA to ethanol. Acetyl-CoA produced from sugar oxidation also generates reduced ferredoxin but there is no known pathway for the transfer of electrons from reduced ferredoxin to NAD in C. bescii. Herein, we engineered a strain of C. bescii using a more stable genetic background than previously reported and heterologously-expressed adhE from Clostridium thermocellum (which grows optimally (Topt) at 60 °C) with and without co-expression of the membrane-bound Rnf complex from Thermoanaerobacter sp. X514 (Topt 60 °C). Rnf is an energy-conserving, reduced ferredoxin NAD oxidoreductase encoded by six genes (rnfCDGEAB). It was produced in a catalytically active form in C. bescii that utilized the largest DNA construct to be expressed in this organism. The new genetic lineage containing AdhE resulted in increased ethanol production compared to previous reports. Ethanol production was further enhanced by the presence of Rnf, which also resulted in decreased production of pyruvate, acetoin and an uncharacterized compound as unwanted side-products. Using crystalline cellulose as the growth substrate for the Rnf-containing strain, 75 mM (3.5 g/L) ethanol was produced at 60 °C, which is 5-fold higher than that reported previously. This underlines the importance of redox balancing and paves the way for achieving even higher ethanol titers in C. bescii.

An undergraduate biochemistry laboratory experiment has been developed using biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance (SPR), in which students obtain and analyze kinetic data for a protein‐protein interaction. Optical biosensing is a technique of choice to determine kinetic and affinity constants for biomolecular interactions. Measurements can be made in real‐time without labels, making biosensing particularly appropriate for the teaching laboratory. In the described exercise, students investigate the kinetics of Protein A‐human Immunoglobin G binding under conditions that mimic simple 1:1 binding. Students prepare appropriate serial dilutions of IgG and set up a microplate for the experiment by aliquotting biotinylated Protein A, buffer, and IgG solutions. A commercial BLI sensor, the FortéBio Octet QK, is used to measure binding. While data are collected students prepare a spreadsheet with which they will simulate the data to determine kon, koff, and KD. Raw data from the sensor are then exported to the spreadsheets for analysis. Optimized experiment timing, regeneration methods and other parameters are described to increase throughput and reduce cost. The experiment is readily adaptable to other biosensing platforms such as SPR instruments. Biochemistry and Molecular Biology Education Vol. 38, No. 6, pp. 400‐407, 2010