Protein Folding—How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry

Abstract

Advanced hydrogen exchange (HX) methodology can now determine the structure of protein folding intermediates and their progression in folding pathways. Key developments over time include the HX pulse labeling method with nuclear magnetic resonance analysis, the fragment separation method, the addition to it of mass spectrometric (MS) analysis, and recent improvements in the HX MS technique and data analysis. Also, the discovery of protein foldons and their role supplies an essential interpretive link. Recent work using HX pulse labeling with MS analysis finds that a number of proteins fold by stepping through a reproducible sequence of native-like intermediates in an ordered pathway. The stepwise nature of the pathway is dictated by the cooperative foldon unit construction of the protein. The pathway order is determined by a sequential stabilization principle; prior native-like structure guides the formation of adjacent native-like structure. This view does not match the funneled energy landscape paradigm of a very large number of folding tracks, which was framed before foldons were known and is more appropriate for the unguided residue-level search to surmount an initial kinetic barrier rather than for the overall unfolded-state to native-state folding pathway.