Triple resonance experiments are a set of multi-dimensional
nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique to observe local magnetic fields around atomic nuclei. The sample is placed in a magnetic fiel ...
(NMR) experiments that link three types of
atomic nuclei
The atomic nucleus is the small, dense region consisting of protons and neutrons at the center of an atom, discovered in 1911 by Ernest Rutherford based on the 1909 Geiger–Marsden gold foil experiment. After the discovery of the neutron ...
, most typically consisting of
1H,
15N and
13C. These experiments are often used to assign specific resonance signals to specific atoms in an isotopically-enriched protein. The technique was first described in papers by
Ad Bax
Adriaan "Ad" Bax (born 1956) is a Dutch-American molecular biophysicist. He was born in the Netherlands and is the Chief of the Section on Biophysical NMR Spectroscopy at the National Institutes of Health. He is known for his work on the methodolo ...
, Mitsuhiko Ikura and
Lewis Kay in 1990,
and further experiments were then added to the suite of experiments. Many of these experiments have since become the standard set of experiments used for sequential assignment of NMR resonances in the determination of
protein structure by NMR. They are now an integral part of solution NMR study of proteins, and they may also be used in
solid-state NMR
Solid-state NMR (ssNMR) spectroscopy is a technique for characterizing atomic level structure in solid materials e.g. powders, single crystals and amorphous samples and tissues using nuclear magnetic resonance (NMR) spectroscopy. The anisotropic pa ...
.
Background
There are two main methods of determining protein structure on the atomic level. The first of these is by
X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
, starting in 1958 when the crystal structure of
myoglobin
Myoglobin (symbol Mb or MB) is an iron- and oxygen-binding protein found in the cardiac and skeletal muscle tissue of vertebrates in general and in almost all mammals. Myoglobin is distantly related to hemoglobin. Compared to hemoglobin, myoglobi ...
was determined. The second method is by NMR, which began in the 1980s when
Kurt Wüthrich
Kurt Wüthrich (born 4 October 1938 in Aarberg, Canton of Bern) is a Swiss chemist/biophysicist and Nobel Chemistry laureate, known for developing nuclear magnetic resonance (NMR) methods for studying biological macromolecules.
Education and ...
outlined the framework for NMR structure determination of proteins and solved the structure of small globular proteins. The early method of structural determination of protein by NMR relied on proton-based homonuclear NMR spectroscopy in which the size of the protein that may be determined is limited to ~10 KDa. This limitation is due to the need to assign NMR signals from the large number of nuclei in the protein – in larger protein, the greater number of nuclei results in overcrowding of resonances, and the increasing size of the protein also broadens the signals, making resonance assignment difficult. These problems may be alleviated by using heteronuclear NMR spectroscopy which allows the proton spectrum to be edited with respect to the
15N and
13C chemical shifts, and also reduces the overlap of resonances by increasing the number of dimensions of the spectrum. In 1990, Ad Bax and coworkers developed the triple resonance technology and experiments on proteins isotopically labelled with
15N and
13C,
with the result that the spectra are dramatically simplified, greatly facilitating the process of resonance assignment, and increasing the size of the protein that may be determined by NMR.
These triple resonance experiments utilize the relatively large magnetic
couplings between certain pairs of nuclei to establish their connectivity. Specifically, the
1J
NH,
1J
CH,
1J
CC, and
1J
CN couplings are used to establish the scalar connectivity pathway between nuclei. The magnetization transfer process takes place through multiple, efficient one-bond magnetization transfer steps, rather than a single step through the smaller and variable
3J
HH couplings. The relatively large size and good uniformity of the one-bond couplings allowed the design of efficient magnetization transfer schemes that are effectively uniform across a given protein, nearly independent of conformation.
Triple resonance experiments involving
31P may also be use for nucleic acid studies.
Suite of experiments
These experiments are typically named by the nuclei (H, N, and C) involved in the experiment. CO refers to the
carbonyl carbon, while CA and CB refer to
Cα and Cβ respectively, similarly HA and HB for Hα and Hβ (see diagram for examples of experiments). The nuclei in the name are ordered in the same sequence as in the path of magnetization transfer, those nuclei placed within parentheses are involved in the magnetization transfer pathway but are not recorded. For reason of sensitivity, these experiments generally start on a proton and end on a proton, typically via
INEPT and reverse INEPT steps. Therefore, many of these experiments are what may be called "out-and-back" experiments where, although not indicated in the name, the magnetization is transferred back to the starting proton for signal acquisition.
Some of the experiments are used in tandem for the resonance assignment of protein, for example HNCACB may be used together with CBCA(CO)NH as a pair of experiments. Not all of these experiments need to be recorded for sequential assignment (it can be done with as few as two), however extra pairs of experiments are useful for independent assessment of the correctness of the assignment, and the redundancy of information may be necessary when there is ambiguity in the assignments. Other experiments are also necessary to fully assign the side chain resonances.
TROSY Transverse relaxation optimized spectroscopy (TROSY) is an experiment in protein NMR spectroscopy that allows studies of large molecules or complexes.
The application of NMR to large molecules is normally limited by the fact that the line widths ge ...
versions of many of these experiments exist for improvement in sensitivity. Triple resonance experiments can also be used in sequence-specific backbone resonance assignment of
magic angle spinning
In solid-state NMR spectroscopy, magic-angle spinning (MAS) is a technique routinely used to produce better resolution NMR spectra. MAS NMR consists in spinning the sample (usually at a frequency of 1 to 130 kHz) at the magic angle θm (ca. 5 ...
NMR spectra in
solid-state NMR
Solid-state NMR (ssNMR) spectroscopy is a technique for characterizing atomic level structure in solid materials e.g. powders, single crystals and amorphous samples and tissues using nuclear magnetic resonance (NMR) spectroscopy. The anisotropic pa ...
.
A large number triple-resonance NMR experiments have been created, and the experiments listed below is not meant to be exhaustive.
HNCO
The experiment provides the connectivities between the amide of a residue with the carbonyl carbon of the preceding residues.
It is the most sensitive of the triple resonance experiments. The sidechains
carboxamides of
asparagine
Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...
and
glutamine
Glutamine (symbol Gln or Q) is an α-amino acid that is used in the biosynthesis of proteins. Its side chain is similar to that of glutamic acid, except the carboxylic acid group is replaced by an amide. It is classified as a charge-neutral, ...
are also visible in this experiment. Additionally, the
guanidino
Guanidine is the compound with the formula HNC(NH2)2. It is a colourless solid that dissolves in polar solvents. It is a strong base that is used in the production of plastics and explosives. It is found in urine predominantly in patients expe ...
group of
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
, which has similar coupling constant to the carboxamide group, may also appear in this spectrum. This experiment is sometimes used together with HN(CA)CO.
HN(CA)CO
Here, the amide resonance of a residue is correlated with the carbonyl carbon of the same residue, as well as that of the preceding residue. The intra-residue resonances are usually stronger than the inter-residues one.
HN(CO)CA
This experiment correlates the resonances of the amide of a residue with the Cα of the preceding residue. This experiment is often used together with HNCA.
HNCA
This experiment correlates the chemical shift of amide of a residue the Cα of the same residue as well as those of the preceding residue.
Each strip gives two peaks, the inter and intra-residue Cα peaks. Peak from the preceding Cα may be identified from the HN(CO)CA experiment which gives only the inter-residue Cα.
CBCA(CO)NH
CBCA(CO)NH, or alternatively HN(CO)CACB, correlates the resonances of the amide of a residue with the Cα and Cβ of the preceding residue. Two peaks corresponding to the Cα and Cβ are therefore visible for each residue. This experiment is normally used together with HNCACB. The sidechain carboxamide of glutamines and asparagines also appear in this spectra in this experiment. CBCA(CO)NH is sometimes more precisely called (HBHA)CBCA(CO)NH as it starts with aliphatic protons and ends on an amide proton, and is therefore not an out-and-back experiment like HN(CO)CACB.
HNCACB
HNCACB, or alternatively CBCANH, correlates the chemical shift of amide of a residue the Cα and Cβ of the same residue as well as those of the preceding residue. In each strip, four peaks may be visible – 2 from the same residue and 2 from the preceding residue. Peaks from the preceding residue are usually weaker, and may be identified using CBCA(CO)NH. In this experiment, the Cα and Cβ peaks are in opposite phase, i.e. if Cα appears as a positive peak, then Cβ will be negative, making identification of Cα and Cβ straightforward. The extra information of Cβ from the CBCA(CO)NH/HNCACB set of experiments makes identification of residue type easier than HN(CO)CA/HNCA, however the HNCACB is a less sensitive experiment and may be unsuitable for some proteins.
The CBCANH experiment is less suitable for larger protein as it is more susceptible to the line-width problem than HNCACB.
CBCACO(CA)HA
This experiment provides the connectivities between the Cα and Cβ with the carbonyl carbon and Hα atoms within the same residue. The sidechain
carboxyl
In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxylic ...
group of aspartate and glutamate may appear weakly in this spectrum.
CC(CO)NH
This experiment provides connectivities between the amide of a residue and the aliphatic carbon atoms of the preceding residue.
H(CCO)NH
This experiment provides connectivities between the amide of a residue and the hydrogen atoms attached to the aliphatic carbon of the preceding residue.
HBHA(CO)NH
This experiment correlates the amide resonance to the Hα and Hβ of the preceding residue.
Sequential assignment
Pairs of experiments are normally used for sequential assignment, for example, the HNCACB and CBCA(CO)NH pair, or HNCA and HNC(CO)CA. The spectra are normally analyzed as strips of peaks, and strips from the pair of experiments may be presented together side by side or as an overlay of two spectra. In the HNCACB spectra 4 peaks are usually present in each strip, the Cα and Cβ of one residue as well as those of its preceding residue. The peaks from the preceding residue can be identified from the CBCA(CO)NH experiment. Each strip of peaks can therefore be linked to the next strip of peaks from an adjoining residue, allowing the strips to be connected sequentially. The residue type can be identified from the chemical shifts of the peaks, some, such as serine, threonine, glycine and alanine, are much easier to identify than others. The resonances can then be assigned by comparing the sequence of peaks with the amino acid sequence of the protein.
References
{{Reflist, colwidth=33em
External links
Triple resonance experiments for proteinsIntroduction to 3D Triple Resonance Experiments
Protein structure
Nuclear magnetic resonance spectroscopy