Pacific Northwest National Laboratory's high magnetic field (800 MHz) NMR spectrometer being loaded with a sample.

Protein NMR is a field within structural biology, that applies NMR spectroscopy to proteins. The goal is to obtain information about the structure and dynamics of the protein(s) under investigation. The field was pioneered by among others the 2002 Nobel laureate Kurt Wüthrich, and is being continually used and improved in both academia and the biotech industry. Structure determination by NMR spectroscopy usually consists of the following phases, each using a separate set of highly specialized techniques: Sample preparation, resonance assignment, restraint generation and structure calculation and validation. Pacific Northwest National Laboratory 800MHz NMR Spectrometer File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Pacific Northwest National Laboratory 800MHz NMR Spectrometer File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Structural biology is a branch of molecular biology concerned with the study of the architecture and shape of biological macromolecules--proteins and nucleic acids in particular—and what causes them to have the structures they have. ... Nuclear Magnetic Resonance Spectroscopy is the name given to the technique which exploits the magnetic properties of nuclei. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The Nobel Prizes (pronounced no-BELL or no-bell) are awarded annually to people who have done outstanding research, invented groundbreaking techniques or equipment, or made outstanding contributions to society. ... Kurt Wüthrich (born October 4, 1938) is a Swiss chemist and Nobel laureate. ... Plato is credited with the inception of academia: the body of knowledge, its development and transmission across generations. ...

Sample preparation

Protein NMR is performed on aqueous samples of highly purified protein. Usually the sample consist of between 300 and 600 µL with a protein concentration in the range 0.1 – 3 mM. The source of the protein can be either natural or produced in an expression system using recombinant DNA techniques (genetic engineering). Recombinantly expressed proteins are usually easier to produce in sufficient quantity, and makes isotopical labelling possible. Protein purification is the process of isolating proteins from a homogenate, which may comprise cell and tissue components, including DNA, cell membrane and other proteins. ... A molar is the fourth kind of tooth in mammals. ... Recombinant DNA is an artificial DNA sequence resulting from the combining of two other DNA sequences in a plasmid. ... An iconic image of genetic engineering; this 1986 autoluminograph of a glowing transgenic tobacco plant bearing the luciferase gene of the firefly strikingly demonstrates the power and potential of genetic manipulation. ... Gene expression (also protein expression or often simply expression) is the process by which a genes information is converted into the structures and functions of a cell. ... Isotopes are forms of an element whose nuclei have the same atomic number–-the number of protons in the nucleus--but different atomic masses because they contain different numbers of neutrons. ...

The most abundant isotopes of carbon and nitrogen, ¹²C and ¹⁴N, do not posses a nuclear spin which is the physical property NMR spectroscopy exploits. Thus NMR of proteins from natural sources are restricted to utilizing NMR based solely on protons However the less common isotopes, ¹³C and ¹⁵N, are suitable for NMR, and therefore labelling the proteins with these compounds open up possibilities for doing more advanced multidimensional experiments (see section on Resonance assignment). Isotopical labelling is done by growing the expression host on a growth media enriched in the desired isotope(s). Since isotopically enriched compounds remains expensive, organisms capable of growing on a defined minimal medium, containing only one ¹³C source (usually glucose, but occasionally glycerol and methanol), and ¹⁵N source ( usually NH₄Cl or (NH₄)₂SO₄). These organisms include bacteria (Escherichia coli is the most frequently used) and unicellular fungi. General Name, Symbol, Number carbon, C, 6 Chemical series nonmetals Group, Period, Block 14, 2, p Appearance black (graphite) colorless (diamond) Atomic mass 12. ... This article does not cite its references or sources. ... For alternative meanings see proton (disambiguation). ... Glucose (Glc), a monosaccharide, is one of the most important carbohydrates. ... ... Methanol, also known as methyl alcohol or wood alcohol, is a chemical compound with chemical formula CH3OH. It is the simplest alcohol, and is a light, volatile, colourless, tasteless, flammable, poisonous liquid with a very faint odor. ... Kingdoms/Phyla/Divisions Actinobacteria Aquificae Bacteroidetes/Chlorobi Chlamydiae/Verrucomicrobia Chloroflexi Chrysiogenetes Cyanobacteria Deferribacteres Deinococcus-Thermus Dictyoglomi Fibrobacteres/Acidobacteria Firmicutes Fusobacteria Gemmatimonadetes Nitrospirae Planctomycetes Proteobacteria Spirochaetes Thermodesulfobacteria Thermomicrobia Thermotogae Iya Kodie Lovie :D ŴÕŤ Ú ÚÞ ²? lol ow good am i :P iya nat lv :D im not lookin forward to d. ... E. coli redirects here. ... A microorganism or microbe is an organism that is so small that it is microscopic (invisible to the naked eye). ... Divisions Chytridiomycota Zygomycota Ascomycota Basidiomycota The Fungi (singular: fungus) are a large group of organisms ranked as a kingdom within the Domain Eukaryota. ...

The purified protein is dissolved in a buffer (optional) and adjusted to the desired solvent conditions. Buffer can have various meanings: In chemistry, the term buffer refers to a buffer solution used to stabilize the pH (acidity) of a liquid. ...

NMR Spectroscopy

For a thorough review of the theory of NMR see the article Nuclear Magnetic Resonance. Pacific Northwest National Laboratorys high magnetic field (800 MHz) NMR spectrometer being loaded with a sample. ...

Data collection

Protein NMR utilizes multidimensional NMR experiments to obtain information about the protein. Ideally, each distinct nuclei in the molecule experiences a distinct chemical environment and thus has a distinct chemical shift by which it can be recognized. The multidimensionality stems from the fact, that through an elaborate pulse sequence, magnetization can be transferred between nuclei, and thus connections between different nuclei can be detected. Additional experimental dimensions may also disperse signals that in lower dimensions may overlap or be indistinguishable. The array of NMR experiments used on proteins fall in two main categories: One where magnetization is transferred through the chemical bonds, and one where the transfer is through space, irrespective of the bonding structure. The first category is used to assign the different chemical shifts to a specific nucleus, and the second is primarily used to generate the distance restraints used in the structure calculation (and in the assignment with unlabelled proten). In nuclear magnetic resonance (NMR), the chemical shift describes the dependence of nuclear magnetic energy levels on the electronic environment in a molecule. ...

Depending on the concentration of the sample, on the magnetic field of the spectrometer, and on the type of experiment, a single multidimensional NMR experiment on a protein sample may take hours or even several days to obtain suitable signal-to-noise ratio through signal averaging, and to allow for sufficient evolution of magnetization transfer through the various dimensions of the experiment. Other things being equal, higher-dimensional experiements will take longer than lower-dimensional experiments.

If the protein is ¹⁵N labelled the NMR phase is usually initiated by recording a 2D HSQC spectrum. In theory the HSQC has one peak for each H bound to an ¹⁵N. Thus the HSQC allows you to evaluate whether the expected number of peaks is present and thus to identify possible problems with multiple conformation or peaks not showing up for various reasons. The relatively quick HSQC experiment helps determine the feasibility of doing subsequent longer, more expensive, and more elaborate experiments. It is not possible to assign peaks to specific atoms from the HSQC alone. Conformation generally means structural arrangement. ...

Resonance Assignment

In order to analyze the NMR data, it is important to get an resonance assignment for the protein. That is to find out which chemical shift in each dimension corresponds to which atom. Several different types of experiments have been invented to achieve this. The procedure depends on whether the protein is isotopically labelled or not, since a lot of the assignment experiments depend on ¹³C and ¹⁵N. In nuclear magnetic resonance (NMR), the chemical shift describes the dependence of nuclear magnetic energy levels on the electronic environment in a molecule. ...

Homonuclear NMR

With unlabelled protein the usual procedure is to record a set of 2D homonuclear NMR experiments: COSY, TOCSY an NOESY. A 2D NMR experiment produces a 2 dimensional spectrum. The unit of the both axis are chemical shift. The cosy and tocsy transfer magnetization through the chemical bonds between adjacant protons. The COSY experiment is only able to transfer magnetisation between protons on adjacent atoms, whereas in the TOCSY experiment the protons are able to relay the magnetization, so it is transferred among all the protons that are connected by adjacent protons. Thus in the COSY, an alpha proton transfers magnetization to the beta proton(s), the beta protons transfers to the alpha and gamma protons (if any present), the gamma proton transfers to the beta and the delta protons etc.. In the TOCSY the alpha (and all the other protons) is able to transfer magnetization to the beta, gamma, delta, epsilon if they are connected by a continuous chain of protons. The continuous chain of protons are the sidechain of the individual amino acids. Thus these two experiments are used to build so called spin systems, that is build a list of resonances of the chemical shift of the peptide proton, the alpha proton(s) and all the protons from each residue’s sidechain. Which chemical shifts corresponds to which nuclei in the spinsystem is determined by the COSY connectivities and the fact that different types of protons (alpha, beta, gamma etc.) have characteristic chemical shifts. To connect the different spinsystems in a sequential order, the NOESY experiment have to be used. Because this experiment transfers magnetization through space, it will show crosspeaks to all protons that are close in space regardless of whether they are in the same spin system or not. The neighbouring residues are inherently close in space, so the assignments can be made by the peaks in the NOESY with other spin systems. In chemistry, an amino acid is any molecule that contains both amino and carboxylic acid functional groups. ... A residue, broadly, is anything left behind by a reaction or event. ...

One important problem using homonuclear NMR is overlap between peaks. That is different protons having the same (or very similar chemical shifts). This problem becomes greater as the protein becomes greater, so homonuclear NMR is usually restricted to small proteins or peptides. In music, especially Schenkerian analysis, an elision, overlap, or rather reinterpretation (Umdeutung), is the perception, after the fact, of a metrically weak final chord (of a chord progression) as being in a strong position as the initial chord of the next progression. ...

¹⁵N NMR

The process of resonance assignment for a ¹⁵N labelled sample is similar to the homonuclear case. No experiment can be performed that transfers magnetisation between two spin systems through bonds either. The main difference is the ability to record ¹⁵N edited 3D experiments: TOCSY N HSQC and NOESY N HSQC. These experiments build onto the HSQC experiment, but have an additional proton dimension. It can be visualised as each peak in the HSQC having the TOCSY/NOESY peaks stacked onto it. Thus if the TOCSY peak from an amide proton, H_N, has a cross peak to its alpha proton, H_alpha, at the coordinates (H_N, H_alpha) in the TOCSY spectrum, the corresponding peak would be at (H_N, H_alpha,N) in the TOCSY N HSQC. Thus it is possible to resolve overlaps in the proton dimension, if the corresponding nitrogens have chemical shifts distinct from one another.

¹³C, ¹⁵N NMR

When the protein is labelled with ¹³C and ¹⁵N it is possible to record an experiment that transfers magnetisation over the peptide bond, and thus connect different spin systems through bonds. This is usually done using some of the following experiments: HNCO, HNCACO, HNCA, HNCOCA, HNCACB and CBCACONH. All six experiments consist of a HSQC plane expanded with a carbon dimension. In the HNCO the spectrum contains peaks at the chemical shifts of the carbonyl carbons in the residue of the HSQC peak and the previous one in the sequence. The HNCACO only contains the one from the previous residue, and it is thus possible to assign the carbonyl carbon shifts that corresponds to each HSQC peak and the one previous to that one. Thus it is possible to make the assignment by matching the shifts of each spin system's own and previous carbons. The HNCA and HNCOCA works similarly, just with the alpha carbons rather than the carbonyls, and the HNCACB and the CBCACONH contains both the alpha carbon and the beta carbon. Usually several of these experiments are required to resolve overlap in the carbon dimension. This procedure is usually less ambiguous than the noesy based method, since it is based on through bond transfer. In the NOESY-based methods additional peaks that are close in space but not belonging to the sequential residues will appear confusing the assignment process. When the sequential assignment has been made it is usually possible to assign the sidechains using HCCH-TOCSY, which is basically a TOCSY experiement resolved in an additional carbon dimension.

Restraint Generation

In order to make structure calculations a number of experimentially determined restraints have to be generated. These fall into different categories, the most widely used is distance restraints and angle restraints.

Distance Restraints

A crosspeak in a noesy experiment, signifies spatial proximity between the two nuclei in question. Thus each peak can be converted in to a maximum distance between the nuclei usually between 1,8 and 6 Å. The intensity of a noesy peak is proportional to the distance to the minus 6th power, so the distance is determined according to intensity of the peak. The intensity-distance relationship is not exact, so usually a distance range is used. Ã…, or Ã¥, is a letter, representing a vowel, in the Swedish, Danish, Norwegian, Walloon, Chamorro and Finnish alphabets. ...

It is of great importance, to assign the noesy peaks to the correct nuclei based on the chemical shifts. If this task is performed manually it is usually very labor intensive, since proteins usually have thousands of noesy peaks. Some computerprograms such as CYANA perform this task automatically, coupled to a structure calculation.

To obtain as accurate assignments as possible it is a great advantage to have access to ¹³C and ¹⁵N noesy experiments, since they help to resolve overlap in the proton dimension. This leads to faster and more reliable assignments, and in turn to better structures.

Angle Restraints

In addition to distance restraints, restraints on the torsion angles of the chemical bonds, typically the psi and phi angles can be generated. One approach is to use the Karplus equation, to generate angle restraints from coupling constants. Another approach uses the chemical shifts to generate angle restraints. Both methods use the fact that the geometry around the alpha carbon affects the coupling constants and chemical shifts, so given the coupling constants and/or the chemical shifts, a qualified guess can be made about the torsion angles. In physics, a coupling constant, usually denoted g, is a number that determines the strength of an interaction. ...

Orientation Restraints

The analyte molecules in a sample can be partially ordered with respect to the external magnetic field of the spectrometer through the introduction of large particles (relative to the size of the molecule under study), such as viruses or bicelles. This partial ordering allows residual dipolar coupling to develop during the experiment between nuclei in the sample, which in turn allow the derivation of additional restraints representing varying relative orientations of different portions of the molecule with respect to a single global reference frame.

NMR structure determination generates an ensemble of structures. The structures will only converge, if the data is sufficient to dictate a specific fold. In these structures, it is only the case for a part of the structure. From PDB 1SSU.

Structure Calculation

The experimentially determined restraints can be used as input for the structure calculation process. That is using computer programs (Such as CYANA or XPLOR) researchers attempt to satisfy as many of the restraints as possible, ind addition to general properties of proteins such as bond lengths and angles. The algorithms convert the restraints and the general protein properties into energy terms, and thus tries to minimize the energy. The process results in an ensemble of structures that, if the data were sufficient to dictate a certain fold, will converge.

Dynamics

In addition to structures, NMR can yield information on the dynamics of various parts of the protein. This usually involves measuring relaxation rates (see article on nuclear magnetic resonance). Pacific Northwest National Laboratorys high magnetic field (800 MHz) NMR spectrometer being loaded with a sample. ...

See Also

NMR NMR may refer to: Nuclear magnetic resonance, a phenomenon involving the interaction of atomic nuclei and external magnetic fields Nielsen Media Research, a U.S. company which measures TV, radio and newspaper audiences This is a disambiguation page — a navigational aid which lists other pages that might otherwise share the...

NMR Spectroscopy Nuclear Magnetic Resonance Spectroscopy is the name given to the technique which exploits the magnetic properties of nuclei. ...

X-ray Crystallography

Structural Biology

Solid State NMR Nuclear magnetic resonance spectroscopy (NMR) characterized by the appearance of anisotropic interactions is referred to as solid-state NMR spectroscopy (SSNMR). ...

References

Protein NMR Spectroscopy : Principles and Practice (1995) John Cavanagh, Wayne J. Fairbrother, Arthur G. Palmer III, Nicholas J. Skelton, Academic Press