Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems

Abstract

Most proteins are only barely stable, which impedes research, complicates therapeutic applications, and makes proteins susceptible to pathologically destabilizing mutations. Our ability to predict the thermodynamic consequences of even single point mutations is still surprisingly limited, and established methods of measuring stability are slow. Recent advances are bringing protein stability studies into the high-throughput realm. Some methods are based on inferential read-outs such as activity, proteolytic resistance or split-protein fragment reassembly. Other methods use miniaturization of direct measurements, such as intrinsic fluorescence, H/D exchange, cysteine reactivity, aggregation and hydrophobic dye binding (DSF). Protein engineering based on statistical analysis (consensus and correlated occurrences of amino acids) is promising, but much work remains to understand and implement these methods.

Figure 1. Principles of screening for protein stability

Most methods of screening for stability modify the unfolded state or observe some unique property of it—for example, by proteolysis, reaction with an exposed cysteine, amide proton exchange, hydrophobic dye binding, or aggregation. Typically, the protein solution is heated or challenged with increasing concentration of chemical denaturant to establish when the signal is observed. Because most HT stability screening methods involve observation of an irreversible reaction, care must be taken in interpreting the data as a change in equilibrium stability. While some methods enable measurements in the presence of other proteins, most require sufficient purification so that other proteins to do produce the unfolding signal.