Split green fluorescent protein as a modular binding partner for protein crystallization.

Thomas C. Terwilliger,Li-Wei Hung,Geoffrey S. Waldo,Todd O. Yeates,Hau B. Nguyen

doi:10.1107/s0907444913024608

Abstract

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

Highlights

Structural characterization of proteins, protein complexes and small molecules is essential to understand cellular functions from enzymology to macromolecular machines
We show here that -strands 10 and 11 of green fluorescent protein (GFP) can be inserted as a hairpin into a protruding loop of a target protein, which when complemented by GFP(1–9) gives rise to a well ordered complex with two polypeptide-chain crossings between the two components which is amenable to crystal structure analysis
The sequences belonging to strands 10 and 11 of superfolder GFP (sfGFP) were pruned from sfGFP and grafted between the original strands 8 and 9 of sfCherry based on the designed constructs (Fig. 4a)

Summary

Introduction

Structural characterization of proteins, protein complexes and small molecules is essential to understand cellular functions from enzymology to macromolecular machines. Carrier proteins have been inserted into loops of transmembrane proteins (Engel et al, 2002) and the insertion of T4 lysozyme into a loop of the 2-adrenergic receptor is an example of a successful application of this strategy (Rosenbaum et al, 2007; Cherezov et al, 2007) Noncovalent crystallization chaperones such as Fab and Fv fragments of antibodies (Kovari et al, 1995; Lange & Hunte, 2002; Lee et al, 2005; Ostermeier et al, 1995; Monroe et al, 2011) and designed ankyrin-repeat protein (DARPin; Monroe et al, 2011) have alternatively been used to produce complexes with target molecules.

Methods

Crystallization

Strategy for modular design

Cherry fluorescent protein as a target protein

Selection of a permissive insertion site in sfCherry

Crystal structure of sfCherry alone