Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

Abstract

Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

Structure determination of a major facilitator peptide transporter: Inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121

“…Initial crystal hits were readily obtained regardless of whether the protein had been purified in DDM, DM or NM, but the best diffraction was attained when using NM, a temperature of 277 K, and a crystallant containing small PEGs as well as a buffer at near neutral to slightly alkaline pH (Table 1). Shifting to a 24-well format and scaling up the drop size was not found beneficial, which is an experience that we have also had with other integral membrane proteins [19,30,31]. For optimization, a wide range of conventional additives e.g.…”

A general protocol for the generation of Nanobodies for structural biology

“…Collective efforts of several laboratories have demonstrated that Nanobodies are exquisite chaperones to crystallize complex biological systems such as membrane proteins 1-3 , transient multiprotein assemblies 2,4-6 , transient conformational states 1 , intrinsically disordered proteins 7,8 or can be used as structural probes of protein misfolding and fibril formation 9,10 . Nanobodies (Nbs) are the small (15kDa) and stable single domain fragments harboring the full antigen-binding capacity of the original heavy chain-only antibodies that naturally occur in Camelids 11,12 .…”

Deep sequencing in library selection projects: what insight does it bring?

“…While such chaperones were originally derived from immunized animals, recombinant display techniques using immunized or naïve binder sources as starting materials has broadened the nature of molecules used to include synthetic recombinant Fabs [5,6], designed ankyrin repeat proteins (DARPINs) [7–9], fibronectin domains [10] and nanobodies [11]. Any method that simplifies the generation of suitable crystallization chaperones is to be welcomed, and it is anticipated that the combination of NGS with display technologies will facilitate the development of effective chaperones, particularly if selection strategies can be specifically designed to select such molecules directly.…”