Using Fluorescence Anisotropy for Ligand Binding Kinetics of Membrane Proteins.

Determining ligand binding kinetics provides an indirect route to probe the functional capabilities of the binding pocket of a membrane protein receptor. Presented in this unit are four ligand-binding protocols that provide data useful for characterizing membrane proteins, including equilibrium binding, thermostability, competitive ligand binding, and kinetic ligand binding. These techniques use fluorescence anisotropy, which is safer, less costly, and simpler to execute than radioactive ligand binding. Each protocol may be used on its own or in combination with others to quantify a number of ligand binding constants. © 2018 by John Wiley & Sons, Inc.

Cutaneous microvascular perfusion responses to insulin iontophoresis are differentially affected by insulin resistance after spinal cord injury.

NEW FINDINGS: What is the central question of this study? What impact does insulin resistance have on cutaneous perfusion responses to insulin iontophoresis in vascular beds with markedly reduced or functionally ablated sympathetic nervous system vasomotor function resulting from spinal cord injury? What is the main finding and its importance? Persons with spinal cord injury have sublesional microvascular endothelial dysfunction, as indicated by a blunted cutaneous perfusion response to acetylcholine iontophoresis, and the presence of insulin resistance has a further confounding effect on endothelium-mediated changes to cutaneous perfusion in the lower extremities. Endothelium-mediated mechanisms that regulate skin blood flow might play an integral role in optimizing skin perfusion in vascular beds with sympathetic nervous system vasomotor impairment, such as in spinal cord injury (SCI). Insulin is a vasoactive hormone and second messenger of nitric oxide that facilitates endothelium-mediated dilatation. The effects of insulin resistance (IR) on sublesional cutaneous perfusion responses to insulin provocation have yet to be described in persons with SCI. Persons with SCI and an able-bodied (AB) cohort were divided into subgroups based upon fasting plasma insulin concentration cut-offs for IR (≥13.13 mIU ml-1 ) or insulin sensitivity (IS; <13.13 mIU ml-1 ), as follows: AB, IS (ABIS, n = 21); SCI, IS (SCIS, n = 21); AB, IR (ABIR, n = 9); and SCI, IR (SCIR, n = 11). Laser Doppler flowmetry characterized peak blood perfusion unit (BPU) responses (percentage change from baseline) to insulin, acetylcholine or placebo iontophoresis in the lower extremities; BPU responses were log10 transformed to facilitate comparisons, and the net insulin response (NetIns) BPU response was calculated (insulin minus placebo BPU response). The NetIns was significantly greater in both IS groups compared with their corresponding IR group. The acetylcholine-mediated BPU responses in the SCI subgroups were significantly lower than those in the ABIS group. The proportional BPU responses of NetIns to acetylcholine in the IS cohorts (i.e. ABIS and SCIS) were significantly greater (P < 0.05) than that of each IR subgroup. The presence of IR has a confounding effect on sublesional microvascular endothelium-mediated cutaneous perfusion responses to provocation. Preservation of endothelial sensitivity to its agonists appears to be an important modifiable risk factor to optimize cutaneous perfusion in the lower extremities of persons with SCI.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 µg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 µg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.