Hydrogen Bond Flexibility and Water Dynamics in the Far Red Fluorescent Protein TagRFP675

Abstract

Next-generation far-red emitting fluorescent proteins (FPs) with enhanced fluorescence quantum yields and high photostability are highly desirable. They would enable deeper tissue penetration and lengthened imaging times given a lower autofluorescence background, lower light scattering, and higher transmission beyond 650 nm. The farthest red emitting FP engineered to date is TagRFP675, which has a 77 nm Stokes shift. The red emission of TagRFP675 is attributed to several H-bonding contacts involving Q41 and S28 at the N-acylimine position with additional H-bonds at the phenoxy end of the chromophore with N143, N158, and R197. We used molecular dynamics (MD) simulations to explore the relationship between flexibility of the chromophore environment and the large Stokes shift in TagRFP675 and related variants mKate and its mutant mKate-M41Q. Both TagRFP675 and mKate-M41Q have large Stokes shift as compared to mKate. Analysis of the hydrogen bonds around the chromophore reveal that in both TagRFP675 and mKate-M41Q have an extended hydrogen bond network connecting Q106-water-F65-Q41-S28 whereas in mKate, this network does not extend beyond F65. The water molecule involved in the hydrogen bond network shows significantly larger flexibility and mobility in TagRFP675 as compared to mKate-M41Q, highlighting the role of the chromophore environment for large bathochromic shifts.