Surveying protein structure and function using bis-arsenical small molecules

Abstract

Exploration across the fields of biology, chemical biology, and medicine has led to an increasingly complex, albeit incomplete, view of the interactions that drive life's processes. The ability to monitor and track the movement, activity, and interactions of biomolecules in living cells is an essential part of this investigation. In our laboratory, we have endeavored to develop tools that are capable not only of monitoring protein localization but also reporting on protein structure and function. Central to our efforts is a new strategy, bipartite tetracysteine display, that relies on the specific and high-affinity interaction between a fluorogenic, bis-arsenical small molecule and a unique protein sequence, conformation, or assembly. In 1998, a small-molecule analogue of fluorescein with two arsenic atoms, FlAsH, was shown by Tsien and coworkers to fluoresce upon binding to a linear amino acid sequence, Cys-Cys-Arg-Glu-Cys-Cys. Later work demonstrated that substituting Pro-Gly for Arg-Glu optimized both binding and fluorescence yield. Our strategy of bipartite tetracysteine display emanated from the idea that it would be possible to replace the intervening Pro-Gly dipeptide in this sequence with a protein or protein partnership, provided the assembled protein fold successfully reproduced the approximate placement of the two Cys-Cys pairs. In this Account, we describe our recent progress in this area, with an emphasis on the fundamental concepts that underlie the successful use of bis-arsenicals such as FlAsH and the related ReAsH for bipartite display experiments. In particular, we highlight studies that have explored how broadly bipartite tetracysteine display can be employed and that have navigated the conformational boundary conditions favoring success. To emphasize the utility of these principles, we outline two recently reported applications of bipartite tetracysteine display. The first is a novel, encodable, selective, Src kinase sensor that lacks fluorescent proteins but possesses a fluorescent readout exceeding that of most sensors based on Förster resonance energy transfer (FRET). The second is a unique method, called complex-edited electron microscopy (CE-EM), that facilitates visualization of protein-protein complexes with electron microscopy. Exciting as these applications may be, the continued development of small-molecule tools with improved utility in living cells, let alone in vivo, will demand a more nuanced understanding of the fundamental photophysics that lead to fluorogenicity, as well as creative approaches toward the synthesis and identification of new and orthogonal dye-tag pairs that can be applied facilely in tandem. We describe one example of a dye-sequence tag pair that is chemically distinct from bis-arsenical chemistry. Through further effort, we expect that that bipartite tetracysteine display will find successful use in the study of sophisticated biological questions that are essential to the fields of biochemistry and biology as well as to our progressive understanding of human disease.

FIGURE 1

Linear tetracysteine display, as described by Tsien and co-workers.^, (a) Cartoon depicting the fluorescent labeling of an engineered protein displaying a linear tetracysteine motif. (b) Chemical structures of FlAsH and ReAsH. (c) The identity of the intervening sequence has a substantial effect on the apparent K_d as well as on the quantum yield (Φ) of the peptide–bis-arsenical complex.

FIGURE 2

Monitoring conformational changes using traditional tetracysteine display. Cartoon depicting the use of ReAsH to (a) monitor changes in protein folding and (b) monitor changes in protein assembly.

FIGURE 3

Bipartite tetracysteine display: initial results. (a) Schematic representation of intramolecular bipartite tetracysteine display. (b) Misfolded bipartite tetracysteine variants of aPP (F24P,Y31P) bind FlAsH and ReAsH (25 nM) with diminished affinities relative to the wild-type bipartite tetracysteine constructs. (c) Schematic representation of intermolecular bipartite tetracysteine display. (d) Monomeric bipartite tetracysteine variants of GCN4 (L20P) bind FlAsH and ReAsH (25 nM) with diminished affinities relative to the wild-type bipartite tetracysteine dimer.

FIGURE 4

Structural dependence of bipartite tetracysteine binding sites. Crystallographic B-factor putty rendering for bipartite tetracysteine mutants of (a) aPP (PDB 2BF9), (b) GCN4 (PDB 2ZTA), (c) p53 (PDB 1TUP), (d) EmGFP (PDB 1EMA), and (e) CRABP-1 (PDB 1CBI). Close up views of the bis-arsenical binding site are shown to the side, along with the corresponding apparent K_d values.

FIGURE 5

Applications of bipartite tetracysteine display. (a) Schematic representation of a Src kinase sensor. A conformational change that occurs upon phosphorylation by Src kinase can be detected using ReAsH. (b) Schematic representation of complex-edited electron microscopy (CE-EM). Irradiation of the ReAsH complex in the presence of diaminobenzidine (DAB) polymerizes the DAB surrounding each protein complex. The EM signal resulting from subsequent treatment with OsO₄ is therefore edited to visualize only those proteins that are closely interacting.

FIGURE 6

Two possible mechanisms for photoinduced electron transfer (PeT) quenching in bis-arsenical fluorophores. (a) Orbital energy diagram depicting a PeT quenching mechanism for bis-arsenical fluorophores. (b) Simplified orbital overlap diagram representing a quenched fluorophore (top) in which the As orbitals are in conjugation with the fluorophore π-system, or an unquenched fluorophore (bottom) in which the As orbitals are orthogonal to the π-system.

FIGURE 7

Development of orthogonal dye–tag pairs. (a) Chemical structure of AsCy3. (b) Cartoon depicting the fluorescent complex that forms when AsCy3 binds to an orthogonal tetracysteine sequence. (c) Chemical structure of RhoBo. (d) Cartoon portraying the fluorescent complex that forms when RhoBo binds to a novel tetraserine sequence.