A monomeric StayGold fluorescent protein

Abstract

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Fig. 1. Structure of the StayGold fluorescent protein at 1.6 Å resolution.

a, Overall structure of the StayGold dimer. b, Top–down view of the StayGold fluorophore and chloride ion. c, Side-on view of the StayGold fluorophore highlighting residues above and below the plane of the fluorophore. d, Secondary structure arrangement in the StayGold peptide. Locations of residues forming the fluorophore are shown in green and residues forming the dimer interface are purple.

Fig. 2. Rational design of a monomeric StayGold derivative for use in biological research.

a, Size-exclusion chromatography showing the oligomeric state of StayGold and its variants. A mixture of StayGold and mStayGold (that is, E138D) is used to demonstrate their separability. b, Location of two monomerizing mutations (E138D and Y187A) and nonmonomerizing (T195K) mutation in the context of the StayGold dimer. c, Close-up view of the StayGold dimer interface. d, Photostability of StayGold and mStayGold relative to GFP. Fluorescence is given as a percentage of the initial brightness of the S. pombe cytokinetic ring for cells expressing the indicated fluorescent protein fusion to the myosin light chain. Error bars indicate s.d. (n = 20). e, Representative images showing photobleaching for S. pombe cytokinetic ring using GFP, StayGold or mStayGold protein fusions. Yellow triangles indicate the cytokinetic ring.