Structure-based design of covalent nanobody binders for a thermostable green fluorescence protein

Abstract

The use of green fluorescence protein (GFP) has advanced numerous areas of life sciences. An ultra-thermostable GFP (TGP), engineered from a coral GFP, offers potential advantages over traditional jellyfish-derived GFP because of its high stability. However, owing to its later discovery, TGP lacks the extensive toolsets available for GFP, such as heavy chain-only antibody binders known as nanobodies. In this study, we report the crystal structure of TGP in complex with Sb92, a synthetic nanobody identified from a previous in vitro screening, revealing Sb92’s precise three-dimensional epitope. This structural insight, alongside the previously characterized Sb44-TGP complex, allows us to rationally design disulfide bonds between the antigen and the antibody for tighter interactions. Using biochemical analysis, we identify two bridged complexes (TGP A18C-Sb44 V100C and TGP E118C-Sb92 S57C), with the TGP-Sb92 disulfide pair showing high resistance to reducing agents. Our study expands the toolkit available for TGP and should encourage its wider applications.

1. Introduction

It is difficult to find a research field of biosciences without the contribution of fluorescence proteins (FPs). In cell biology, FPs allow researchers to pinpoint protein locations [1–3], observe protein-protein interactions [4–6], and track biological processes at the cellular [7], tissue [8], organ [9], and whole-body levels [10]. In biochemistry, FPs enable convenient characterization of fusion partners, including their expression and purification yield [11] and stability [12–16], without requiring pure samples. This is particularly beneficial for challenging-to-purify proteins, such as membrane proteins. FPs can also be engineered as reporters for cellular activities. For example, fusion proteins that combine circularly permutated green FP (GFP), calmodulin, and its binding peptide can track intracellular calcium changes by linking Ca2+-induced binding with improved GFP folding [17]. Split FPs are widely used to detect protein-protein interactions [18], monitor inter-compartment protein translocation [19], and tether protein termini for crystallization [20,21]. Split GFP variants with an inverted topology between the two halves have been used as sensors to detect specific protease activities, such as caspases, within cells [22,23].

As a class, FPs are among the most extensively engineered proteins. The jellyfish GFP, for example, has undergone modifications to improve brightness, maturation speed, folding efficiency, and color variation [24]. A particularly interesting GFP variant, Azami Green [25], originates from coral and has attracted considerable attention because of its unique properties. Azami Green shares only 27% sequence identity with the jellyfish GFP, containing a Gln-Tyr-Gly chromophore that is different from that of the jellyfish GFP (Ser/Thr-Tyr-Gly). Nevertheless, both retain a similar β-barrel structure known as a β-can [26]. Owing to its homology with red fluorescent proteins, Azami Green was the first GFP to be engineered to emit red fluorescence [27]. By applying consensus mutagenesis, the Bradbury group created a variant named CGP (consensus green protein), which is expressed with higher yield and brightness than Azami Green [28]. Through recursive cycles of introducing and removing destabilizing elements combined with directed evolution, the same group developed an extremely stable CGP variant called eCGP123, which retains full fluorescence even after overnight heating at 80°C [29]. Additionally, eCGP123 maintains over 80% fluorescence in 6 M guanidine hydrochloride (GuHCl), a chaotropic condition typically disruptive to secondary structures. To address its tendency to aggregate, structure-guided mutations led to an even more stable variant, TGP (thermostable green protein) [26], which retains fluorescence after 50 days at 85°C. The melting temperature (Tm) of TGP is approximately 95°C (with 20 min of heating), which is approximately 20°C higher than that of the superfold GFP [15].

TGP has proven to be a superior fusion partner for membrane proteins, offering expression levels up to 30 times higher than those of conventional GFP fusions in widely used systems such as Escherichia coli, Saccharomyces cerevisiae, baculovirus, and mammalian cells [15]. Its extreme stability also enables thermostability assessments of fusion membrane proteins across a wide temperature range using fluorescence-detection size-exclusion chromatography. In this approach, the fluorescen...