The low stability of natural proteins often limits their use in therapeutic, industrial and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for determining the approximate stabilities of protein variants at high throughput and low cost. The method is based on binding to a hydrophobic dye akin to ANS, which fluoresces upon binding to molten globules and thermal denaturation intermediates. No inherent properties of the protein, such as enzymatic activity or presence of an intrinsic fluorophore, are required. Very small sample sizes are analyzed using a realtime PCR machine, enabling the use of high-throughput purification. We show that the apparent T M values obtained from HTTS are approximately linearly related to those from CD thermal denaturation for a series of four-helix bundle hydrophobic core variants. We demonstrate similar results for a small set of TIM barrel variants. This inexpensive, general and scaleable approach enables the search for conservative, stable mutants of biotechnologically-important proteins, and it provides a method for statistical correlation of sequence-stability relationships.Natural proteins are often too unstable for therapeutic or industrial applications, or even for crystallography or directed evolution experiments. 1 There is still no reliable way to predict stabilizing mutations, and biophysical characterization of proteins is generally large-scale and low-throughput. 2 Except for enzymes, where enzymatic activity can be screened at elevated temperatures, high-throughput methods of screening for stability are lacking. Notably, the dominant classes of protein drugs-hormones, antibodies, cytokines, etc.-are binding proteins or ligands, not enzymes. Here we demonstrate that a dye-binding thermal shift screen, an extension of the ThermoFluor® method of screening for protein-ligand interactions, 3 reports the relative thermal stabilities of libraries of protein variants. We call the method HighThroughput Thermal Scanning, or HTTS.In ThermoFluor®, samples of a receptor protein are mixed with an analyte ligand and a fluorescent hydrophobic dye akin to 1-anilinonaphthalene-8-sulphonic acid (ANS). Folded proteins exclude these types of dyes, but molten globules and thermal denaturation intermediates bind them, resulting in a sharp increase in fluorescence. Binding of a ligand to the folded state of the receptor shifts the apparent melting temperature higher, which can be observed by heating the sample in a fluorimeter. Besides for drug discovery, this method has been applied to optimization of ligand and buffer conditions for crystallography. 4 We wished to invert the format of the screen, instead using a library of protein variants under the same conditions of dye and buffer, to probe the approximate relative thermal stabilities of the mutants. Since dye binding is so phy...

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Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which “hidden correlations” can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.

Most proteins are only barely stable, which impedes research, complicates therapeutic applications, 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.

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