Hydrogen-deuterium exchange
Encyclopedia
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 amide
s 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. Hydrogen exchange was first shown and explored by Kaj Ulrik Linderstrøm-Lang.
hydrogens in the peptide bond
s of proteins exchange protons with the solvent
. By changing the solvent from H2O to D2O, deuterons will be incorporated in the amide positions and the exchange reaction can be followed. Most often, deuterium is added to a protein in H2O by diluting the H2O solution with D2O (e.g. tenfold). Usually exchange is performed at physiological pH (7.0–8.0) where proteins are in their most native ensemble of conformational states. See also .
Because the exchange reaction can be either acid
or base
catalyzed, it is strongly pH
dependent. For the backbone amide hydrogens, the minimum exchange rate occurs at approximately pH 2.6, on average. By performing the exchange at neutral pH and then rapidly changing the pH, the exchange rates of the backbone amide hydrogens can be dramatically slowed, or quenched. The pH at which the reaction is quenched depends on the analysis method. For detection by NMR, the pH may be moved to around 4.0–4.5. For detection by mass spectrometry, the pH is dropped to the minimum of the exchange curve, pH 2.6. In the most basic experiment, the reaction is allowed to take place for a set time before it is quenched.
The deuteration pattern of a quenched protein can be stably maintained in aprotic environments. However, analysis of the deuteration is usually performed in an aqueous solution, which means that exchange will continue at a slow rate even after the reaction is quenched. Reversion of deuterated positions after the quench step is referred to as back-exchange and various methods have been devised to correct for this.
and mass spectrometry
. Each of these methods have their advantages and drawbacks.
and deuterium
nuclei
are grossly different in their magnetic properties. Thus it is 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 is difficult to obtain information about amides, that exchange in shorter time frames.
The deuterium nucleus is twice as heavy as the hydrogen nucleus because it contains a neutron
as well as a proton. Thus a protein that contains some deuterium will be heavier than one that contains all hydrogen. As a protein is increasingly deuterated, the molecular mass
increases correspondingly. Detecting the change in the mass of a protein upon deuteration was made possible by modern protein mass spectrometry, first reported in 1991 by Katta and Chait .
The location and relative amount of deuterium exchange along the peptide backbone can be determined roughly by subjecting the protein to proteolysis after the exchange reaction has been quenched. Individual peptides are then analyzed for overall deuteration of each peptide fragment. Using this technique the resolution of deuterium exchange is determined by the size of the peptides produced during digestion . Pepsin
, an acid protease
, is commonly used for proteolysis, as the quench pH must be maintained during the proteolytic reaction. To minimize the back-exchange, proteolysis and subsequent mass spectrometry analysis must be done as quickly as possible. HPLC
separation of the peptic digest is often carried out at low temperature just prior to electrospray mass spectrometry to minimize back-exchange. More recently, UPLC has been used due to its superior separation capabilities .
It was proposed in 1999 that it might be possible to achieve single-residue resolution by using collision-induced dissociation
fragmentation of deuterated peptides in conjunction with tandem mass spectrometry
. It was soon discovered that CID causes "scrambling" of the deuterium position within the peptides . However, fragmentation produced by electron capture dissociation
and electron transfer dissociation
proceed with little or no scrambling under the correct experimental conditions . This suggests that eventually it may be possible to obtain single-residue resolution of H/D exchange reactions on a routine basis.
structure slows exchange. Exchange rates are a function of two parameters: solvent accessibility and hydrogen bonding. Thus an amide which is part of an intramolecular hydrogen bond
will exchange slowly if at all, while an amide on the surface of protein hydrogen bonded to water will exchange rapidly. Amides buried from the solvent but not hydrogen bonded may also have very slow exchange rates. Because both solvent accessibility and hydrogen bonding contribute to the rate of exchange, it becomes difficult to attribute a given exchange rate to a structural element without x-ray crystallography
or NMR structural data.
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 has been used 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 may be protected in the complex and exchange slowly. However, one must bear in mind that H-D exchange cannot be used to locate binding interfaces for all protein-protein interactions. Some protein-protein interactions are driven by electrostatic forces of side chains and are unlikely to change the exchange rate of backbone amide hydrogens, particularly if the amide hydrogens are located in stable structural elements such as alpha helicies.
Lastly, H/D exchange can be used to monitor conformational changes in proteins as they relate to protein function. If conformation is altered as result of post-translational modification, enzyme activation, drug binding or other functional events, there will likely be a change to H/D exchange that can be detected.
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
in which a covalently bonded hydrogen
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
atom is replaced by a deuterium
Deuterium
Deuterium, also called heavy hydrogen, is one of two stable isotopes of hydrogen. It has a natural abundance in Earth's oceans of about one atom in of hydrogen . Deuterium accounts for approximately 0.0156% of all naturally occurring hydrogen in Earth's oceans, while the most common isotope ...
atom, or vice versa. Usually the examined protons are the amide
Amide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...
s in the backbone of a protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. The method gives information about the solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
accessibility of various parts of the molecule, and thus the tertiary structure
Protein structure
Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...
of the protein. Hydrogen exchange was first shown and explored by Kaj Ulrik Linderstrøm-Lang.
The exchange reaction
In solution, AmideAmide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...
hydrogens in the peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...
s of proteins exchange protons with the solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
. By changing the solvent from H2O to D2O, deuterons will be incorporated in the amide positions and the exchange reaction can be followed. Most often, deuterium is added to a protein in H2O by diluting the H2O solution with D2O (e.g. tenfold). Usually exchange is performed at physiological pH (7.0–8.0) where proteins are in their most native ensemble of conformational states. See also .
Because the exchange reaction can be either acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...
or base
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...
catalyzed, it is strongly pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
dependent. For the backbone amide hydrogens, the minimum exchange rate occurs at approximately pH 2.6, on average. By performing the exchange at neutral pH and then rapidly changing the pH, the exchange rates of the backbone amide hydrogens can be dramatically slowed, or quenched. The pH at which the reaction is quenched depends on the analysis method. For detection by NMR, the pH may be moved to around 4.0–4.5. For detection by mass spectrometry, the pH is dropped to the minimum of the exchange curve, pH 2.6. In the most basic experiment, the reaction is allowed to take place for a set time before it is quenched.
The deuteration pattern of a quenched protein can be stably maintained in aprotic environments. However, analysis of the deuteration is usually performed in an aqueous solution, which means that exchange will continue at a slow rate even after the reaction is quenched. Reversion of deuterated positions after the quench step is referred to as back-exchange and various methods have been devised to correct for this.
Detection of H/D exchange
H-D exchange was measured originally by the father of hydrogen exchange Kaj Ulrik Linderstrøm-Lang using density gradient tubes. In modern times, H/D exchange has primarily been monitored by the methods: NMR spectroscopyProtein nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins. The field was pioneered by Richard R. Ernst and Kurt Wüthrich, among others...
and mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
. Each of these methods have their advantages and drawbacks.
NMR spectroscopy
HydrogenHydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
and deuterium
Deuterium
Deuterium, also called heavy hydrogen, is one of two stable isotopes of hydrogen. It has a natural abundance in Earth's oceans of about one atom in of hydrogen . Deuterium accounts for approximately 0.0156% of all naturally occurring hydrogen in Earth's oceans, while the most common isotope ...
nuclei
Atomic nucleus
The nucleus is the very dense region consisting of protons and neutrons at the center of an atom. It was discovered in 1911, as a result of Ernest Rutherford's interpretation of the famous 1909 Rutherford experiment performed by Hans Geiger and Ernest Marsden, under the direction of Rutherford. The...
are grossly different in their magnetic properties. Thus it is possible to distinguish between them by NMR spectroscopy
NMR spectroscopy
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained...
. 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
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....
-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
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
. Because it takes minutes to hours to record the spectra, it is difficult to obtain information about amides, that exchange in shorter time frames.
Mass spectrometry
Mass spectrometry has several advantages over NMR with respect to analysis of H/D exchange reactions: Much less material is needed, the concentration of protein can be very low (as low as 0.1 uM), the size limit is much greater, and data can usually be collected and interpreted much more quickly.The deuterium nucleus is twice as heavy as the hydrogen nucleus because it contains a neutron
Neutron
The neutron is a subatomic hadron particle which has the symbol or , no net electric charge and a mass slightly larger than that of a proton. With the exception of hydrogen, nuclei of atoms consist of protons and neutrons, which are therefore collectively referred to as nucleons. The number of...
as well as a proton. Thus a protein that contains some deuterium will be heavier than one that contains all hydrogen. As a protein is increasingly deuterated, the molecular mass
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
increases correspondingly. Detecting the change in the mass of a protein upon deuteration was made possible by modern protein mass spectrometry, first reported in 1991 by Katta and Chait .
The location and relative amount of deuterium exchange along the peptide backbone can be determined roughly by subjecting the protein to proteolysis after the exchange reaction has been quenched. Individual peptides are then analyzed for overall deuteration of each peptide fragment. Using this technique the resolution of deuterium exchange is determined by the size of the peptides produced during digestion . Pepsin
Pepsin
Pepsin is an enzyme whose precursor form is released by the chief cells in the stomach and that degrades food proteins into peptides. It was discovered in 1836 by Theodor Schwann who also coined its name from the Greek word pepsis, meaning digestion...
, an acid protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
, is commonly used for proteolysis, as the quench pH must be maintained during the proteolytic reaction. To minimize the back-exchange, proteolysis and subsequent mass spectrometry analysis must be done as quickly as possible. HPLC
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
separation of the peptic digest is often carried out at low temperature just prior to electrospray mass spectrometry to minimize back-exchange. More recently, UPLC has been used due to its superior separation capabilities .
It was proposed in 1999 that it might be possible to achieve single-residue resolution by using collision-induced dissociation
Collision-induced dissociation
In Mass spectrometry, Collision-induced dissociation , referred to by some as collisionally activated dissociation , is a mechanism by which to fragment molecular ions in the gas phase. The molecular ions are usually accelerated by some electrical potential to high kinetic energy and then allowed...
fragmentation of deuterated peptides in conjunction with tandem mass spectrometry
Tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.-Tandem MS instruments:...
. It was soon discovered that CID causes "scrambling" of the deuterium position within the peptides . However, fragmentation produced by electron capture dissociation
Electron capture dissociation
Electron-capture dissociation is a method of fragmenting gas phase ions for tandem mass spectrometric analysis . ECD involves the direct introduction of low energy electrons to trapped gas phase ions...
and electron transfer dissociation
Electron transfer dissociation
Electron-transfer dissociation is a method of fragmenting ions in a mass spectrometer. Similar to electron-capture dissociation, ETD induces fragmentation of cations by transferring electrons to them. It was invented by Donald F. Hunt, Joshua Coon, John E. P...
proceed with little or no scrambling under the correct experimental conditions . This suggests that eventually it may be possible to obtain single-residue resolution of H/D exchange reactions on a routine basis.
Applications to protein structure
It is not possible to determine the structure of a protein with H/D exchange nor is it possible to define secondary structural elements. The reasons for this are related to the way in which protein structureProtein structure
Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...
structure slows exchange. Exchange rates are a function of two parameters: solvent accessibility and hydrogen bonding. Thus an amide which is part of an intramolecular hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
will exchange slowly if at all, while an amide on the surface of protein hydrogen bonded to water will exchange rapidly. Amides buried from the solvent but not hydrogen bonded may also have very slow exchange rates. Because both solvent accessibility and hydrogen bonding contribute to the rate of exchange, it becomes difficult to attribute a given exchange rate to a structural element without x-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
or NMR structural data.
H/D exchange has been used to characterize the folding
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
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 has been used 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 may be protected in the complex and exchange slowly. However, one must bear in mind that H-D exchange cannot be used to locate binding interfaces for all protein-protein interactions. Some protein-protein interactions are driven by electrostatic forces of side chains and are unlikely to change the exchange rate of backbone amide hydrogens, particularly if the amide hydrogens are located in stable structural elements such as alpha helicies.
Lastly, H/D exchange can be used to monitor conformational changes in proteins as they relate to protein function. If conformation is altered as result of post-translational modification, enzyme activation, drug binding or other functional events, there will likely be a change to H/D exchange that can be detected.