A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Abstract

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

“…Nevertheless, this GSDMB_C failed to crystallize. Therefore, we produced the GSDMB_C linked to maltosebinding protein (MBP) using the pMALX vectors (34). These recombinant proteins contain the interdomain linker region (Met220-Lys240) that was systematically truncated by 4-to 5-aa segments.…”

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

“…The first approach is based on the observation that both T4L and MBP have been successfully used to crystallize otherwise difficult proteins, including membrane proteins and amyloid proteins. 10,[57][58][59][60][61] The mutation pairs characterized in this study could be readily used to increase the chance of getting crystals. The crystal packing arrangements of such fusion proteins would depend on surface properties of the target protein and would therefore likely be different from the structures presented here.…”

Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms of AIM2 Autoinhibition and Inflammasome Assembly

“…The MBP tag harbors mutations (D82A/ K83A/E172A/N173A/K239A) designed to enhance its crystallization propensity (35,36). Transformed BL21(DE3) Codon Plus RIPL cells (Stratagene, Santa Clara, CA) were grown at 37°C, induced with 0.3 mM isopropyl 1-thio-␤-D-galactopyranoside at 18°C for 4 h, harvested, and lysed in buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl) plus 5 mM imidazole, DNase (Biomatik, Wilmington, DE), and protease inhibitors (Roche Applied Science).…”