Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

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Protein stability: a crystallographer's perspective.

The present application provides the compounds of formula (J), or pharmaceutically acceptable salts, isomers, or a mixture thereof, wherein n, m, q, A', W1, W2, W3, R1, R2, and R3 are described herein. The compounds are inhibitors to the activities of phosphatidylinositol 3-kinase (PI3K) and are useful for treating conditions mediated by one or more PI3K isoforms. The present application further provides pharmaceutical compositions that include a compound of formula (J), or pharmaceutically acceptable salts, isomers, tautomer, or mixture thereof, and methods of using these compounds and compositions for treating conditions mediated by one or more PI3K isoforms.

Phosphatidylinositol 3-kinase inhibitors

Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.2863

Crystal structures of MBP fusion proteins.

Protein-protein interactions (PPIs) control many important physiological processes within human cells. Apoptosis or programmed cell death is closely regulated by pro- and antiapoptotic signals. Dysregulation of this homeostasis is implicated in tumorigenesis and acquired resistance to treatments. The emerging importance of Mcl-1 protein in chemotherapeutic resistance makes it a high priority therapeutic target. Targeting PPIs associated with Mcl-1 presents many challenges for the design of inhibitors. This review focuses on the characterization of the Mcl-1 hot-spots which are related to four hydrophobic pockets P1-P4 and one major electrostatic interaction. Analysis of structural data highlights the high importance of the P2/P3 pockets for the binding of nonpeptide ligands. In order to guide medicinal chemists into making more selective and potent Mcl-1 inhibitors, the Mcl-1 protein is compared to other antiapoptotic proteins.

Hot-Spots of Mcl-1 Protein.

Crystallization chaperones have been used to facilitate the crystallization of challenging proteins. Even though the maltose-binding protein (MBP) is one of the most commonly used crystallization chaperones, the design of optimal expression constructs for crystallization of MBP fusion proteins remains a challenge. To increase the success rate of MBP-facilitated crystallization, a series of expression vectors have been designed with either a short flexible linker or a set of rigid helical linkers. Seven death domain superfamily members were tested for crystallization with this set of vectors, six of which had never been crystallized before. All of the seven targets were crystallized, and their structures were determined using at least one of the vectors. Our successful crystallization of all of the targets demonstrates the validity of our approach and expands the arsenal of the crystallization chaperone toolkit, which may be applicable to crystallization of other difficult protein targets, as well as to other crystallization chaperones.

Design of an expression system to enhance MBP-mediated crystallization

Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.

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Guoping Xu

Papers

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1.