Abstract
Studies of small proteins that exhibit noncooperative, gradual (un)folding can offer unique insights into the rarely accessible intermediate stages of the protein folding processes. Detailed experimental characterization of these intermediate states requires approaches that utilize multiple site-specific probes of the local structure. Isotopically edited infrared (IR) spectroscopy has emerged as a powerful methodology capable of providing such high-resolution structural information. Labeling of selected amide carbonyls with 13C results in detectable side-bands of amide I′ vibrations, which are sensitive to local conformation and/or solvent exposure without introducing any significant structural perturbation to the protein. Incorporation of isotopically labeled amino acids at specific positions can be achieved by the chemical synthesis of the studied proteins. We describe the basic procedures for synthesis of 13C isotopically edited protein samples, experimental IR spectroscopic measurements and analysis of the site-specific equilibrium thermal unfolding of a small protein from the temperature-dependent IR data.
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This work was supported by the National Science Foundation (NSF) grant CAREER 0846140.
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Kubelka, G.S., Kubelka, J. (2022). Multi-Probe Equilibrium Analysis of Gradual (Un)Folding Processes. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_9
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DOI: https://doi.org/10.1007/978-1-0716-1716-8_9
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