Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold

Abstract

The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

“…S3 A-C). At pH 8.0, the 10 nM K d III value for HS1 is identical to the K d III values previously reported for Cyt b 562 (13) and CG6 (18). HS1-ferrous heme dissociation constants, K d II values, were too tight to measure by direct titration and are less than 1 nM.…”

“…HS1 was adapted from a previously reported EGFP-Cyt b 562 integral fusion protein, CG6, that exhibits >99% efficient FRET between EGFP and heme (18). mKATE2 was appended to the N terminus of CG6 with a GlySer linker.…”

“…The genes for mKATE2 and CG6 used to construct HS1 were purchased from GENESCRIPT and codon-optimized for expression in E. coli and S. cerevisiae or humans. Standard published methods for protein expression and purification (18), heme binding studies (20,27), fluorimetry, UV/visible spectroscopy, microscopy, flow cytometry, immunoblotting (41), catalase activity (34), oxygen consumption (41), total heme analysis (42), and cell growth are outlined in SI Appendix, SI Materials and Methods.…”

Random Single Amino Acid Deletion Sampling Unveils Structural Tolerance and the Benefits of Helical Registry Shift on GFP Folding and Structure

“…The absence of InDel mutagenesis as part of the routine protein engineering toolbox is partly due to the difficultly in predicting the local and global structural influence of altering the protein backbone; dogma suggests such mutations are likely to be detrimental due to, for example, disruptive registry shifts in organized secondary structure and perturbing folding pathways (Pascarella and Argos, 1992; Shortle and Sondek, 1995). Consequently, there have been relatively few studies concerning the structural impact of engineered InDel mutations (Arpino et al., 2012a; Heinz et al., 1993; O’Neil et al., 2000; Stott et al., 2009; Vetter et al., 1996), especially regarding how any beneficial effects are exerted at the molecular level (Arpino et al., 2012a). Most of these studies have focused on site-directed introduction of InDels, so little information is available on the general tolerance of proteins to InDels and their beneficial effect.…”

Photophysics, Dynamics, and Energy Transfer in Rigid Mimics of GFP-based Systems

“…Recently, we have demonstrated a method for utilizing a MOF as a multifunctional system for efficient chromophore coupling to replicate the highly efficient ET (∼100%) 26 achieved in the protein system consisting of a variant of the green fluorescent protein (EGFP) and cytochrome b 562 (cyt b 562 , Scheme 1). 27 The major challenge in these studies was not only the achievement of a high efficiency of ET but also the replication of the EGFP photophysical response.…”