Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution. Study holds ProTherm entries: 22067, 22068, 22069, 22070, 22071, 22072, 22073, 22074, 22075, 22076 Extra Details: equilibrium intermediate state, high-energy intermediates, R-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin.
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:53 p.m.
|Number of data points||14|
|Proteins||Cytochrome c ; Fatty acid-binding protein, intestinal ; Alpha-lactalbumin ; Cytochrome c ; Myoglobin ; Alpha-lactalbumin ; Myoglobin ; Fatty acid-binding protein, intestinal|
|Assays/Quantities/Protocols||Experimental Assay: m pH:5.8, ionic:KCl: 20 mM, buffers:Sodium acetate: 20 mM ; Experimental Assay: dG_H2O pH:5.8, ionic:KCl: 20 mM, buffers:Sodium acetate: 20 mM ; Experimental Assay: m pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: dG_H2O pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: m buffers:Tris: 20 mM, pH:7.4, ionic:: ; Experimental Assay: dG_H2O buffers:Tris: 20 mM, pH:7.4, ionic:: ; Experimental Assay: m pH:7.5, ionic:KCl: 100 mM, buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM ; Experimental Assay: m pH:7.4, ionic:: , buffers:Sodium phosphate: 20 mM, prot_conc:10-20 microM|
|Libraries||Mutations for sequence AFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNKFTVKESSNFRNIDVVFELGVDFAYSLADGTELTGTWTMEGNKLVGKFKRVDNGKELIAVREISGNELIQTYTYEGVEAKRIFKKE ; Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE ; Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL ; Mutations for sequence GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG|