Israel M. Scott

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin.

Using interferometry‐based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately 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 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 Ca2+ binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca2+ dependence of CaM binding. Hence, changes in the Ca2+ 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 Ca2+ sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.

Optical biosensing: Kinetics of protein A‐IGG binding using biolayer interferometry

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

Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of ‘open’ conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP+ and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP+ and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP+ shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a ‘conformational lockdown’ model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes. A note to the reader This manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.