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To Exchange or Not to Exchange?

Michael Goshe

Michael Goshe
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To Exchange or Not to Exchange? That is the question — at least for the graduate students participating in our Proteins Journal Club this semester (my apologies to those members of the Shakespearean Journal Club — although I do like the musical version of Hamlet as performed on Gilligan’s Island).





As you may have surmised since this is a biochemistry blog, the topic is hydrogen exchange (HX) in which an amide proton (N-H) of a protein is exchanged for a deuteron (D) where kch is the chemical exchange rate constant and is a function of pH and temperature.

HD exchange reaction

HD exchange reaction

The propensity of an amide proton of an amino acid residue of a protein to deuterium exchange is related to its hydrogen bonding environment. If the amide proton is participating in a strong hydrogen bond with a carbonyl oxygen as occurs in an alpha-helix or beta-sheet, it will be more difficult to exchange with a deuteron than an amide proton that is part of a loop or a region of a protein that has a high degree of flexibility and exposure to solvent (Figure 1). The sites of exchange can be monitored by NMR or mass spectrometry analysis, thus allowing protein folding and structural dynamics to be measured by the accumulation of deuterium.

Principles of HXThe HX chemistry can be conceptualized as spray painting an exposed surface (i.e., the amide nitrogens of proteins susceptible to HX) as shown in Figure 2. However the next, and more difficult step, is to determine where the paint is (N-H vs. N-D) without smearing or rubbing it off (i.e., the conversion of N-D back to N-H during analysis which is known as “back exchange”).

During our first PJC meeting this semester, I presented an introduction to HX and used protein folding as my example. It is interesting to note that although the pioneering work of protein folding by Christian B. Anfinsen began in the 1960s, the use of HX by Kai U. Linderstrøm-Lang and his colleagues at the Carlsberg Laboratories in Copenhagen preceded that work by nearly 10 years. Linderstrøm-Lang wanted to experimentally confirm Linus Pauling’s work on the presence of helices and sheets in proteins by measuring the hydrogen bonded structures by HX. He quantified deuterium by a mass technique that measured the density of tiny water droplets to about 1 ppm using a density gradient column. Linderstrøm-Lang also inferred the basic dynamic mechanisms involved in protein HX processes and wrote the equations for measuring HX.

Since every amino acid residue in a protein has an amide proton, then every residue can be probed using HX. However, being able to monitor HX exchange at every residue is dependent upon the complexity of the system being studied and the technique being used to measure the exchange. NMR is an effective way to monitor residue resolution regarding sites of exchange, but very large proteins systems are precluded by this approach, thus requiring the use of liquid chromatography-tandem mass spectrometry (LC/MS/MS). Due to the variety of mass spectrometry instrumentation and the gains in sensitivity and scanning speeds, the field of HX-MS has developed into a technique that is now poised to provide some impressive insights into complex protein folding mechanisms and dynamic interaction phenomena between large protein complexes. Case in point is the paper I presented on the kinetics of protein folding. This study was conducted by the lab of S. Walter Englander where they used their HX-MS platform to characterize the kinetic folding mechanism of the large two-domain maltose binding protein (MBP), a protein of 370 residues. The authors on this paper did an exemplary job of fine tuning their system to minimize back exchange (i.e., the loss of label due to sample workup and/or LC/MS/MS analysis) and implemented some novel data analysis tools developed in their group to obtain structural resolution of the folding process of the largest protein studied by HX-MS by over 100 residues. In my opinion, this paper will be looked upon as a seminal work in the field of protein folding in particular and HX-MS in general and should usher a new era regarding the use of HX-MS to study larger dynamic protein systems. So take a few minutes to check out this paper on your own — it will be worth it.

Conceptualization of HXIn the days to follow, our Proteins Journal Club will explore HX on a variety of platforms and proteins systems and these will be posted in future blogs. So it is not “Alas poor HX! I knew it, Horatio…”, but instead we eagerly anticipate acquainting ourselves with HX and hope to learn more about it — at least as well as we can via a journal club setting — and perhaps this may inspire some of us to venture out and implement the technique in our own research.




1. Walters B.T., Mayne L., Hinshaw J.R., Sosnick T.R. & Englander S.W. (2013). Folding of a large protein at high structural resolution, Proceedings of the National Academy of Sciences, 110 (47) 18898-18903. DOI: