Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

• Published in: Cell systems Vol. 12; no. 6; pp. 547 - 560
• Main Authors: Atsavapranee, Beatriz, Stark, Catherine D., Sunden, Fanny, Thompson, Samuel, Fordyce, Polly M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.06.2021
• Subjects: biophysical characterization; calorimetry; mass spectrometry; protein folding; protein folding kinetics; protein folding thermodynamics; protein stability; proteolysis; spectroscopy; calorimetry; spectroscopy; mass spectrometry; protein folding; protein folding thermodynamics; biophysical characterization; protein folding kinetics; protein stability; proteolysis
• ISSN: 2405-4712; 2405-4720; 2405-4720
• DOI: 10.1016/j.cels.2021.05.009

Folding a linear chain of amino acids into a three-dimensional protein is a complex physical process that ultimately confers an impressive range of diverse functions. Although recent advances have driven significant progress in predicting three-dimensional protein structures from sequence, proteins are not static molecules. Rather, they exist as complex conformational ensembles defined by energy landscapes spanning the space of sequence and conditions. Quantitatively mapping the physical parameters that dictate these landscapes and protein stability is therefore critical to develop models that are capable of predicting how mutations alter function of proteins in disease and informing the design of proteins with desired functions. Here, we review the approaches that are used to quantify protein stability at a variety of scales, from returning multiple thermodynamic and kinetic measurements for a single protein sequence to yielding indirect insights into folding across a vast sequence space. The physical parameters derived from these approaches will provide a foundation for models that extend beyond the structural prediction to capture the complexity of conformational ensembles and, ultimately, their function. •Linking protein sequence to stability and function is critical for medicine and industry•Protein sequence/stability relationships can be visualized as folding landscapes•Systematic quantitative stability measurements allow mapping of folding landscapes•Emerging high-throughput technologies enable stability measurements at scale Atsavapranee and Stark et al. review methods for measuring protein stability at a variety of resolutions and throughputs, including calorimetry, spectroscopy, mass spectrometry, gel electrophoresis, and sequencing approaches. The authors highlight benefits and limitations of each method and discuss why quantitative and systematic measurements are essential for understanding the relationship between protein sequence, stability, and function.