Everything about Hydrogen-deuterium Exchange totally explained
Hydrogen-deuterium exchange (also called H-D or H/D exchange) is a
chemical reaction in which a covalently bonded
hydrogen atom is replaced by a
deuterium atom, or vice versa. Usually the examined protons are the
amides in the backbone of a
protein. The method gives information about the
solvent accessibility of various parts of the molecule, and thus the
tertiary structure of the protein.
The exchange reaction
Amide protons in the
peptide bonds of proteins exchange with the protons from the
solvent, when in an aqueous solution. Thus by changing the solvent from H
2O to D
2O, the exchange reaction can be followed. The exchange reaction can either be catalyzed by
acid or
base, and is thus strongly
pH dependent. For peptide groups, the minimum
reaction rate occurs at roughly pH 2.2. Rapidly changing solution pH to approximately 2.2 is known as
quenching, and is usually combined with rapid freezing to stop the reaction. The deuteration pattern of a quenched protein isn't completely stable, so usually the detection has to happen as soon as possible. H-D exchange that occurs after quenching is known as
back-exchange, and various methods have been devised to correct for this. Usually the reaction is performed by diluting the H
2O solution with D
2O (for example tenfold). The reaction is then allowed to take place for a set time, and the reaction is quenched. If the reaction is to be detected with mass spectrometry, the protein is usually digested with
pepsin before analysis. Pepsin is most active at low pH, which makes it optimal for cleavage under quench conditions.
Detection of H/D exchange
In modern times H/D exchange has primarily been monitored by the methods:
NMR spectroscopy and
mass spectrometry. Each of these methods have their advantages and drawbacks.
NMR spectroscopy
Hydrogen and
deuterium nuclei are grossly different in their magnetic properties. Thus it's possible to distinguish between them by
NMR spectroscopy. Typically
HSQC spectra are recorded at a series of timepoints while the hydrogen is exchanging with the deuterium. Since the HSQC experiment is specific for hydrogen, the signal will decay exponentially as the hydrogen exchanges. It is then possible to fit an exponential function to the data, and obtain the exchange constant. This method gives
residue-specific information for all the residues in the protein simultaneously. The major drawback is that it requires a prior assignment of the spectrum for the protein in question. This can be very labor intensive, and usually limits the method to proteins smaller than 25
kDa. Because it takes minutes to hours to record the spectra, it's difficult to obtain information about amides, that exchange in shorter time frames.
Mass spectrometry
The deuterium nuclei is heavier than the proton, which means that the
molecular mass of the protein increases as hydrogens are exchanged for deuterium. Originally, the progress of the H/D exchange of a molecule was estimated by the resulting change in its
density. This approach has been updated using the more sophisticated and accurate method of mass spectrometry. Mass spectrometry allows determination of the molecular mass with great precision, and for smaller peptides it can easily distinguish between molecular weight differences of 1 Da. Typically the protein is digested by
pepsin before analysis by mass spectrometry to distinguish between the different regions of the protein. Thus the resultion is typically limited by the amount of pepsin cleavage sites in the protein. It has been proposed, that it's possible to achieve residue resultion by using fragmentation of the peptides by
tandem mass spectrometry. This method is being complicated by extensive hydrogen/deuterium positional exchange during the mass analysis, which renders this method useless. Mass spectrometry has several advantages over NMR: Much less material is needed and it doesn't require as high a
solubility, the size limit is much greater and the data is usually much faster to
assign, especially with the use of tandem mass spectrometry. More details can be found at http://www.hxms.com.
Applications to protein structure
The basic assumption in the analysis of H/D exchange data is that the exchange rate reflects the exposure of the particular amide to the solvent. Thus an amide buried in the
hydrophobic core of a protein will exchange slowly if at all, while an amide on the surface will exchange rapidly.
H/D exchange has been used to characterize the
folding pathway of proteins, by refolding the protein under exchange conditions. The parts of the structure that form rapidly, will be protected quickly, and thus not exchanged, whereas areas that fold late in the pathway will be exposed to the exchange for longer periods of time. Thus H/D exchange can be used to determine the sequence of various folding events. The critical factor determining the time resolution of this approach is the time required for quenching.
H/D exchange is used widely to characterize protein-protein interactions. The exchange reaction needs to be carried out with the isolated proteins and with the complex. The exchanging regions are then compared. If a region is buried by the binding, the amides in this region will be protected in the complex and exchange slowly. Thus the method reveals the interaction interface. Subsequently the data can be used as input for a computer
docking simulation, to build a computer model of the complex.
This application of H-D exchange was pioneered by
Kaj Ulrik Linderstrøm-Lang.
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