A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions.
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.
Dai
Susie Y
SY
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Fitzgerald
Michael C
MC
D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D017666:Deuterium Oxide; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D017510:Protein Folding; D013816:Thermodynamics; D014508:Urea
D050556:Fatty Acid-Binding Proteins; D014508:Urea; D045304:Cytochromes c; D007768:Lactalbumin; D017666:Deuterium Oxide; D019791:Guanidine