The 2.0 Å Crystal Structure of a Pocilloporin at pH 3.5: The Structural Basis for the Linkage Between Color Transition and Halide Binding

Abstract

The pocilloporin Rtms5 and an engineered variant Rtms5H146S undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), approximately 453 nm) transitions of Rtms5 and Rtms5H146. The pK(a) for the blue-yellow transition of Rtms5H146S increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 angstroms resolution the crystal structure of a yellow form of Rtms5H146S at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5H146S structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5H146S and binding to halides.

A Structural Basis for the pH-dependent Increase in Fluorescence Efficiency of Chromoproteins

“…Increased fluorescence emission for Rtms5 and Rtms5 H146S was not observed at acidic pH. 29 In order to investigate this observation in detail, we determined the effect of alkaline pH on the fluorescence and absorption spectra of Rtms5 H146S . Fluorescence emission at the λ max em (Figure 1(a) and inset) was enhanced ∼ 20-fold above pH 10.5, showing a maximum at ∼ pH 11.0 and thereafter decreased to almost zero emission at pH 11.5.…”

“…The increase in Φ F observed in several proteins at similar pH suggests a common mechanism, as would occur if the site were conserved. The chromophore is anionic at pH > 4 29 indicating that the titration does not occur on the chromophore. Computational pK a estimates indicated two candidates, Glu215 ( Figure 7) and Cys105 (data not Figure 5.…”

“…The increased fluorescence efficiency of HcRed has been attributed to the presence of a coplanar chromophore conformation, whilst the low fluorescence efficiency of several chromoproteins including Rtms5 and its variants has been attributed to a trans non-coplanar chromophore. 5,7,11,29 Importantly, the chromophore in DsRed and EqFP611 are coplanar. 8,19 The chromophore in a photoconverted variant of AsFP595 although having undergone trans-cis isomerization remains non-coplanar leaving some doubt as to the nature of the fluorescent species in such proteins kindled by intense illumination.…”

A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

“…Acylimine linkages are susceptible to nucleophilic attack, and when present in FPs undergo addition of water across the double bond when the protein is exposed to extremes of pH. Acylimine hydration results in a reduction in the extent of conjugation of the chromophore, and a characteristic blue-shift in its absorbance spectra [10], [17], [20]. Rtms5 Y67F or Rtms5 Y67F/H146S were incubated in buffer at pH 2.3, and their absorbance spectra determined at selected time points.…”

Theoretical Studies of Green and Red Fluorescent Proteins

“…This has led to a series of publications [51][52][53][54][56][57][58] that have elaborated both key structural and mechanistic issues relating to the RFP HcRed [57] and pocilloporin Rtms5 H146S [59,60] in recent years, and provides an important experimental context within which the knowledge gained from our calculations can aid in furthering experimental development of new engineered proteins. In this regard simulations and modeling can add fundamental understanding to the direction aspect of a directed evolution approach, since the physical interactions and mechanisms introduced by mutations that yield improved RFPs are unknown, and mutagenesis studies alone cannot reveal them.…”