An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Abstract

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.

Crystal structures of Dronpa complexed with quenchable metal ions provide insight into metal biosensor development

“…Particularly, the amino acid sequence of His210 and His212 ( i and i + 2) on the β12‐strand of Dronpa was identical to that previously reported in robust transition metal binding in a β‐sheet mode . Many FPs have shown Cu 2+ ‐induced fluorescence quenching , but the configuration of the Cu 2+ binding was still unknown. Thus, determination of the Cu 2+ ‐binding site in Dronpa could be useful to understand other Cu 2+ sensitive FPs.…”

“…The FRET‐specific signals of fluorescent proteins (EBFP2, mCeruleans3, iq‐mEmerald, mVenus, mApple and mKate2) revealed an average distance of 12.2 Å (distance range was approximately 7–19 Å) between the metal ions and the chromophores . The distances of the metal ions from the chromophore for Dronpa are within the range of the distances for the other metal‐bound FPs determined by FRET measurements . The overall structures showed similarities with the apo‐Dronpa structure (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2GX2) with r.m.s.…”

“…A previously published study measured the distances of intact energy transfer between the quenchable metal ions (FRET donor) and the chromophores (FRET accept) by using FRET measurements . The FRET‐specific signals of fluorescent proteins (EBFP2, mCeruleans3, iq‐mEmerald, mVenus, mApple and mKate2) revealed an average distance of 12.2 Å (distance range was approximately 7–19 Å) between the metal ions and the chromophores . The distances of the metal ions from the chromophore for Dronpa are within the range of the distances for the other metal‐bound FPs determined by FRET measurements .…”

“…In iq‐mEmerald, the two engineered surface‐exposed histidine residues in a β‐sheet ( i and i + 2) created a binding site for transition metals . Cu 2+ ion induced the quenching of the fluorescence intensity, whereas other metal ions generated little reduction (Co 2+ ) or enhancement (Zn 2+ and Ni 2+ ) of the fluorescence activity . Iq‐mEmerald crystal structures complexed with Ni 2+ and Zn 2+ ions showed that both metal ions interact with engineered histidine residues, consisting of indirect interactions of metal ions with the chromophore .…”

“…Cu 2+ ion induced the quenching of the fluorescence intensity, whereas other metal ions generated little reduction (Co 2+ ) or enhancement (Zn 2+ and Ni 2+ ) of the fluorescence activity . Iq‐mEmerald crystal structures complexed with Ni 2+ and Zn 2+ ions showed that both metal ions interact with engineered histidine residues, consisting of indirect interactions of metal ions with the chromophore . Although these structures provide a framework to help understand the metal‐binding site and fluorescence effects, indirect interactions between quenchable metal ions and chromophores have not been determined yet.…”