Protein stability: a crystallographer's perspective

Abstract

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.

Keywords: crystallizability; protein crystallization; protein disorder; protein stability.

Figure 1

Factors influencing protein stability. (a) Protein compositional stability and conformational stability as key determining factors for successful crystallization. The stability properties of the protein determine whether the process of crystal formation is possible. Thermodynamics establish the necessary conditions for crystallization, and the kinetics and dynamics of the processes determine whether a possible scenario actually becomes reality. Only if all of the parameters are satisfied will crystal formation proceed. Figure adapted from Rupp (2015 ▸). (b) The marginal net stability of a folded protein is highlighted with respect to the contributing factors; the overwhelming lack of conformational stability is only marginally balanced by the contribution of van der Waals (VdW), hydrogen-bonding (H-bonds) and hydrophobic forces. Figure adapted from http://bit.ly/1L921Oi.

Figure 2

Matrix of examples of protein stability and disorder. (a) Examples of proteins with high conformational stability include the protein–protein destabilizing compound cyclosporin in complex with calcineurin and cyclophilin (Huai et al., 2002 ▸) and (b) the protein–protein stabilizing drug Tafamidis in combination with transthyretin (Bulawa et al., 2012 ▸). (c) Examples of proteins with low conformational stability include the serpins, which undergo large changes in fold and oligomerization state (Yamasaki et al., 2008 ▸), and (d) intrinsically disordered proteins (IDPs) such as the tumor suppressor protein p53 (Mujtaba et al., 2004 ▸).

Figure 3

HSQC spectrum of folded, unstructured and apo and ligand-bound proteins. (a) Two-dimensional ¹H–¹⁵N heteronuclear single-quantum coherence (HSQC) NMR spectrum showing the distinct discrimination in the region below 8.3 p.p.m. in ω₁ identifying a folded protein (red, sharp peak contours) compared with the wide and unresolved peaks for disordered protein sample (blue contours). Image courtesy of Simon Colebrook, Department of Biochemistry, Oxford University and Joanne Nettleship, Oxford Protein Production facility. (b) HSQC spectrum of apo and ligand-bound protein. The two-dimensional ¹H–¹⁵N HSQC NMR spectrum of bacterial methionine aminopeptidase (bMAP) with (right) and without (left) a tightly bound novel inhibitor (Evdokimov et al., 2007 ▸). Note the drastic improvement in the discrimination of the spectrum for the bMAP–ligand complex compared with the apoprotein. The crystals of the bMAP–ligand complex diffracted to 0.9 Å resolution. Image courtesy of Artem Evdokimov, Procter & Gamble Pharmaceuticals, Mason, Ohio, USA. Figure adapted from Rupp (2015 ▸).

Figure 4

Thermofluor assay of protein melting temperature in the presence of stabilizing ligands. (a) Example melting curves for a protein of unknown function from Eubacterium siraeum (ZP_02421384.1) in the presence of various adenosine- and ribose-containing ligands (grey), adenosine diphosphate (ADP, green), adenosine triphosphate (ATP, red) and control sample with no ligand (blue dashed line). ADP and ATP result in a shift in melting temperature (ΔT _m) of 3 and 8°C, respectively. (b) Matrix of 72 proteins of unknown function screened in a Thermofluor assay against a panel of 327 ligands. The ΔT _m is indicated by the size and color of the data points ranging from 10 to 70°C. Data kindly provided by Anna Grezchnik of the Joint Center for Structural Genomics (JCSG).

Figure 5

Deuterium-exchange mass spectrometry (DXMS) analysis. DXMS was used to guide the construct design used for determining the crystal structure of a putative ethanolamine-utilization protein from Salmonella typhimurium. (a) The left side of the figure shows that the N-terminal portion of the protein is more disordered, or unstructured in solution, as the backbone amide protons are more susceptible to exchange. Deuterium-labelled proteolytic fragments are highlighted in red. (b) The right side of the figure shows that the C-terminal portion of the protein is more ordered, or structured in solution, as the backbone-amide protons are less susceptible to exchange. The region selected for truncation is denoted by a blue arrow. (c) Ribbon diagram of the final crystal structure determined for residues 98–229 showing the compact and ordered structure (loops are shown in green, α-helices in red and β-­strands in yellow; PDB entry 2pyt; Joint Center for Structural Genomics, unpublished work). (d) The same structure colored according to the B-factor value, highlighting the stable core of the protein in blue and the more flexible outer regions in green through red. Data kindly provided by Scott Lesley of the Joint Center for Structural Genomics (JCSG).