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Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

The discovery of hydrogen sulfide (H2S) as a novel gasotransmitter for cell signaling and other pathophysiological processes has spurred tremendous interest in developing analytical methods for its detection in biological systems. Herein, we report the development of a highly responsive and selective genetically encoded H2S probe, hsGFP, for the detection of H2S both in vitro and in living mammalian cells. hsGFP bestows a combination of favorable properties, including large fluorescence responses, high efficiency in folding and chromophore formation, and excellent sensitivity and selectivity toward H2S. As a genetically encoded probe, hsGFP can be readily and precisely localized to subcellular domains such as mitochondria, cell nuclei, and ion channels. hsGFP was further utilized to image H2S enzymatically produced from l-cysteine in human embryonic kidney (HEK) 293T cells.

Peroxynitrite is a highly reactive molecule involved in cell signaling and pathological processes. We hereby report a novel genetically encoded probe, pnGFP, which can selectively sense peroxynitrite. A boronic acid moiety was site-specifically introduced into circularly permuted fluorescent proteins. By examining different protein templates followed with site-targeted random mutagenesis, we identified a selective peroxynitrite sensor, which is essentially unresponsive to other common cellular redox signaling molecules. The new probe has been genetically introduced into mammalian cells to image peroxynitrite at physiologically relevant concentrations.

The detection of hydrogen sulfide (H(2)S), a toxic gas and an important biological signaling molecule, has been a long-time challenge. Here we report genetically encoded fluorescent protein (FP)-based probes that can selectively detect H(2)S. By expanding the genetic codes of E. coli and mammalian cells, FP chromophores were modified with the sulfide-reactive azide functional group. These structurally modified chromophores were selectively reduced by H(2)S, resulting in sensitive fluorescence enhancement detectable by spectroscopic and microscopic techniques. Exploration of a circularly permuted FP led to an improved sensor with faster responses, and the feasibility of using such a genetically encoded probe to monitor H(2)S in living mammalian cells has also been demonstrated.