Implementation of Selective Heteronuclear 2D J-Resolved Method on Standard NMR Spectrometers and Selective Proton Pulse Calibration

Some currently used methods for determining the long-range carbon-proton coupling constants are discussed. We chose the selective heteronuclear 2D J-resolved method because of its good sensitivity and resolution in Fi dimension. We implemented the method on a standard NMR spectrometer with transmitter working at fixed power and no facilities for shaped pulse generation. Selective proton pulse was achieved by using suitable attenuators for the decoupler output power attenuation. The selective proton pulse was calibrated by the indirect method for resonance observation, which allows precise determination of the pulse length. The proposed methodology can be applied to any NMR spectrometer provided that the decoupler output power can be switched by computer at least between two levels in few ,usec. The implementation was tested on a sample of 5-Me-8-OH-quinoline, for which long-range carbon-proton coupling constants have not yet been determined

Review: The future of cystatin engineering

Plant cystatins are naturally occurring protease inhibitors that prevent proteolysis by papain-like cysteine proteases. Their protective action against environmental stresses has been relatively well characterised. Still, there is a need to greatly improve both potency and specificity based on the current rather poor performance of cystatins in biotechnological applications. Research in creating more potent and specific cystatins, including amino acid substitutions in either conserved cystatin motifs and/or at variable amino acid sites, is reviewed. Existing gaps for better understanding of cystatin-protease interactions are further explored. Current knowledge on multi-cystatins or hybrid protease inhibitors involving cystatins as an additional option for cystatin engineering is further outlined along with the nuances of how cystatins with rather unusual amino acid sequences might actually help in cystatin engineering. Finally, future opportunities for application of cystatins are highlighted which include applications in genetically modified transgenic plants for environmental stress protection and also as nutraceuticals, as part of more nutritious food. Further opportunities might also include the possible management of diseases and disorders, often associated with lifestyle changes, and the most immediate and promising application which is inclusion into plant-based recombinant protein production platforms.International Foundation of Science (IFS grant C/5151-1), the NRF Incentive funding for rated researchers (90779) and the NRF National Bioinformatics Functional Genomics program(86947). Funding received from the Genomic Research Institute (GRI), University of Pretoria.http://www.elsevier.com/locate/plantsci2017-05-31hb2016Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil SciencePlant Scienc