Contents 1. Introduction 4025 2. Current Applications of Protein Immobilization Technologies 4026 2.1. Protein Microarrays 4026 2.1.1. Research and Discovery 4027 2.1.2. Proteomic Profiling and Diagnostics 4028 2.2. Biosensors from Immobilized Proteins 4028 2.3. Immobilized Enzymes in Biotechnology and Chemical Manufacturing Processes4029 2.4. Nanotechnology and Single-Molecule Enzymology 4030 3. Chemical and Physical Methods of Protein Immobilization 4030 3.1.

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a “cycle of knowledge” strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

Imbalance of signals that control cell survival and death results in pathologies, including cancer and neurodegeneration. Two pathways that are integral to setting the balance between cell survival and cell death are controlled by lipid-activated protein kinase B (PKB)/Akt and Ca 2؉ . PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca 2؉ stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca 2؉ release through inositol 1,4,5-trisphosphate receptor (InsP3R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP3Rs, significantly reducing their Ca 2؉ release activity. Moreover, phosphorylation of InsP3Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca 2؉ flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKB on InsP3R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKB-mediated abolition of InsP3-induced Ca 2؉ release may afford tumor cells a survival advantage.signaling ͉ cell death ͉ cancer P rotein kinase B (PKB) is a central player in regulating many signaling pathways controlling cell metabolism, growth, and survival (1, 2). PKB elicits these effects by phosphorylating and regulating the activity of downstream targets such as glycogen synthase kinase 3␤ and Bad, or via transcription factors such as Forkhead (1, 3). Because of this critical role of PKB, gain or loss of function is manifest in major disease phenotypes such as cancer and type 2 diabetes (1, 4-6).Ca 2ϩ released from the endoplasmic reticulum (ER) through inositol 1,4,5-trisphosphate (InsP 3 ) receptors (InsP 3 Rs) plays a key role in regulating physiological processes (7). However, under pathological conditions, InsP 3 -induced Ca 2ϩ release (IICR) can be subverted to promote cell death pathways (8-10). The importance of IICR in cell death is underlined by the uncovering of functional interactions with a number of proteins with known proapoptotic and antiapoptotic activity. Notable among these are Bcl-2, Bcl-X L , and cytochrome c (11)(12)(13)(14). PKB has also recently been shown to phosphorylate the InsP 3 R, with consequences for cell survival (15).We investigated whether cross-talk between the phosphatidylinositol 3-kinase (PI3K)/PKB and InsP 3 /Ca 2ϩ signaling pathways regulated how cells responded to death-inducing stimuli. We determined that PKB-mediated phosphorylation of InsP 3 R results in a decrease in the magnitude of IICR and resultant flux of Ca 2ϩ from the ER to mitochondria. Moreover, we show that this decrease in Ca 2ϩ flux caused by PKB-mediated phosphorylation of InsP 3 Rs contributes to protection from the effects of apoptotic stimuli. This prosurvival action of PKB was also apparent in a glioblastoma cell line (U87) that exhibits increase...

Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44 131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.