An Overview of Protein NMR Spectroscopy - Doing Science (2023)

Spectroscopy is "the study of the study and measurement of the absorption and emission of light or electromagnetic radiation by matter as a function of its wavelength and frequency"^[8]🇧🇷 Simply put, it is the study of the interaction of light with a matter or substance.

Based on the nature of the interaction between energy and material, spectroscopy is divided into subcategories including: absorption spectroscopy, emission spectroscopy, elastic reflection and scattering spectroscopy, impedance spectroscopy, resonance spectroscopy, and nuclear spectroscopy.^[13].

Spectroscopy is also divided according to the type of radiant energy and the type of interacting material.

This article discusses the principle, operation and applications of protein NMR spectroscopy. But before we get to that, let's take a quick look at the basics of NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy techniques are used to study the molecular structure of chemical compounds by exploiting the magnetic field around their atomic nuclei. This is done by determining the different local electronic environments of atoms such as hydrogen, carbon, chlorine, etc. into a chemical compound.

It is the only method that generates the three-dimensional structure of the molecule in the solution phase. The electromagnetic resonance phenomenon that occurs provides detailed information about the structure, dynamics, reaction state and chemical environment of molecules such as proteins.

You can read more about terms, advantages and disadvantages of NMR spectroscopy in the article "Spectroscopic nuclear magnetic resonance.“

NMR spectroscopy works on the principle of changing the energy state and orientation of an atomic nucleus in a magnetic field. The change depends on the presence or absence of electrons around the atomic nuclei of a molecule.

This phenomenon pulls all atomic nuclei in the same direction and is called a resonance state. And the energy transfer and the state of the atomic nuclei are indicated by the different signal levels on the screen.

Furthermore, signals from NMR spectra are processed and the amount of energy needed to bring atomic nuclei to the higher state is calculated. Several machines are needed to produce the structure of the molecule in solution.

The formula for calculating the amount of energy required to bring atomic nuclei to their highest state is:

𝚫E = hv

wo =change energy

h = Planck's constant

v = frequency of light

The NMR operating frequency is calculated^[4]:

v = ( 𝜸 /2π ) * second_Ö

Onde 𝜸 =Gyromagnetic relationship that differs in different nuclei.

Bo = the strength of the magnetic field applied by NMR

This equation means that the higher the frequency of light, the greater the applied magnetic field strength. The frequency is therefore directly proportional to the strength of the applied magnetic field.

Resonance spectra are obtained from the chemical shifts of nuclei. Different atomic nuclei show different chemical shifts in the presence of magnetic fields due to the shielding effect of surrounding electrons. This causes the nuclei to resonate at slightly different frequencies.

The formula^[4]used to calculate this:v = ( 𝜸 /2π ) * second_Ö* (1-𝛔)

here𝛔is the shielding effect.

NMR spectroscopy has revealed many mysteries about the behavior of molecules. NMR spectroscopy of proteins helps to study conformational changes, denaturation and internal mobility of proteins, pH titration of individual ionizable amino acid side chains in active sites of enzymes, observation of hydrogen bonded iminos of protons in tRNA, study of paramagnetic centers in metalloproteins etc.^[1].

This method measures the short distances and angles between different protons and then computationally derives the structure of the protein. Modern protein spectroscopy requires multidimensional experiments with¹H,¹³C,y^fifteenN-nuclei and isotopically labeled proteins^[1].

The NMR spectroscopy apparatus consists of nine main parts, a sample holder, magnetic coils, a permanent magnet, a scanning generator, a radio frequency transmitter, a radio frequency detector, a recorder and a reading system. All these parts organize their functions to achieve the desired results.

• Before analyzing protein structure, make sure the protein sample is in a purified form.
• Dissolve the protein in 0.5 mL of water and adjust the pH and ionic strength of the solution. (pH range 3-5)^[1].
• The protein concentration of the sample should be between 1 and 6 mM (ideally between 3 and 6 mM). This is to ensure that a 15-30 mg protein sample is available for NMR spectroscopy.^[1].
• If the molecular weight of the protein is around 12,000, isotopic labeling should be added to the preparation^fifteenN bzw¹³C^[1].

Some points must be considered before sample preparation. Helps remove contamination and reduces background noise during experimentation.

• Carefully avoid/remove paramagnetic contaminants by minimizing sample contact with metal surfaces and equipment. These impurities degrade the spectrum.
• Solution components such as buffer and salt must be prepared using high quality reagents.
• Avoid vigorously shaking the sample as this can lead to protein denaturation.

To determine the structure of the protein in which thousands of resonances will be present,¹The H NMR spectrum overlaps and is crowded. To avoid this confusion, a2-D or 3-D NMR experimentIt is preferable to collect the necessary information.

The 2D and 3D NMR experiments distribute the spectra along two and three frequency axes. The spectra represent "cross peaks" where the spectrum signal is equal on both sides of a diagonal.

The cross peaks arise from magnetization transfer and show that the two nuclei are coupled and have different chemical shifts. The change is so small that it is reported in parts per million (ppm). (You can consult the article1H NMR chemical shiftfor more information on chemical changes during NMR experiments)

The frequency of the detected signal is proportional to the externally applied magnetic field. However, external magnetization is also disturbed and neutralized by the reaction of external electrons. This change in the effective magnetic field causes the spin frequency of the nucleus to change. This is called a "chemical transformation".^[10].

Thus, the effective magnetic field rotating the core is calculated using the formula:

B_Effective= second_applied-B_local

B_Effective= the magnetic field acting on the nuclei.

B_applied= the externally applied magnetic field by NMR.

B_local= the local magnetic field applied by electrons around atomic nuclei.

The most important information is collected byNuclear Overhauser Enhancement (NOE) - Spectroscopyor NOESY. Helps determine the orientation of atoms in a molecule. When the distance between the two protons is about 5.0 Å^Öthen a cross peak between two hydrogen atoms is observed.

Therefore, the NOESY experiment depends on the distance in space. In several 2-D NMR experiments that support this¹H NMR assignment, cross peak means interproton binding ratio. This means that the protons are not separated by more than three covalent bonds and are part of the same amino acid.

In this step, the observed peaks (chemical shifts) in the spectrum are assigned to specific atoms. Resonance mapping helps characterize protein structure and dynamics by specifying the atomic identity of unique frequencies in the spectrum.^[3]🇧🇷 But a protein contains many amino acid residues and this creates thousands of peaks in the spectrum.

This confusion is resolved usingsequential allocation strategy^[3]. Este método é baseado no conhecimento prévio da sequência de aminoácidos e envolve o uso de (homonucleares)¹H NMR experiments. Conventional methods include the heteronuclear or triple resonance approach.

Los¹The H NMR procedure follows three steps^[9]for the resonance assignment:

• Identify the amino acid spin side chain:COZY (correlation spectroscopy) or TOCSY (total correlation spectroscopy) is used for this.^[5]🇧🇷 These experiments generate the spectra of individual amino acids in the protein. Identify the specific pattern of cross peaks to specify the amino acid.
  For example, consider the cross peak patterns of the amino acids aspartate and valine.^[9].

• Gather the identified amino acids from the protein: This step requires NOESY data. A strong NOE indicates the adjacent amino acid and this is due to the presence of a short interproton distance between adjacent amino acid residues. The less intense peaks, defined byme, me+2, jI, i+3,are seen in the secondary structure^[9].
• Combine the results of steps a and b: Combining the above two steps leads to specific and precise resonance assignments that help generate an accurate protein structure.^[9].

To deduce the structure of the protein, it is necessary to determine all three boundary conditions. containsDistance constraints, angle constraints, and orientation constraints🇧🇷 The distance constraint is determined by the NOESY experiment and various software such as PASD, XPLOR-NIH, CYANA and UNIO are used to read the cross peaks.

Resonance assignments can be a cumbersome process when done manually. The use of software algorithms makes the process fast and smooth and also ensures efficient NOESY spectral analysis.

Angular constraints, such as psi () and phi (𝝓) angles, are determined by the Karplus equation or using chemical shift spectra. The twist angle of the molecule can be determined by calculating the coupling constant and the chemical shift.

After all structural constraints, chemical changes and resonance assignment are determined, the structure of the protein is determined using the data. The data obtained helps researchers identify local helical structures, beta sheets and tight turns in the amino acid sequence. The secondary structure obtained is used as a model or computer methods are used to interpret the tertiary structure of the protein.

Current methods for calculating structure from NMR data include:Initial metric matrix spacing geometry followed by molecular dynamics calculations and a variable objective function algorithm, complemented by molecular mechanical energy minimization^[1].

Figure:The first structure of a globular protein called Bull Seminal Proteinase Inhibitor IIA (BUSI IIA) is determined using solution NMR spectroscopy.

Source:Wuthrich Kurt (1990). Determination of the structure of proteins in solution by NMR spectroscopy.Das Journal of Biological Chemistry, 265(36),22059-22062

The process of determining the structure of large proteins weighing around 25 kDa is complex, and an efficient structure cannot be obtained by NMR spectroscopy alone.^[6].

A large number of resonances, an increase in linewidth and a low signal-to-noise ratio lead to clustering and overlapping of spectra. Therefore, advanced isotope labeling and multidimensional experiments proved useful in solving the problem.^[12].

Recent developments have introduced a number of techniques that help weaken magnetization relaxation and refine protein structure.^[11]🇧🇷 These techniques are optimized transverse relaxation spectroscopy (TROSY) and deuteration.

1. In vivo NMR experiments are used to study specific enzyme reaction kinetics in situ and to understand metabolic pathways.^[2].
2. It is a powerful tool for determining solution structure, molecular dynamics and protein folding. It is also used in drug detection and design, and chemical and metabolite analysis.^[fifteen].
3. It is used to identify the structure of proteins, amino acids, organic compounds, carotenoids and the mobility of water in food. It also helps to characterize the geographic origin and ripening time of fruits and vegetables.^[7].
4. It is used by chemists and biochemists to study the properties of organic molecules.^[14].
5. Can be used to infer the structure of an unknown chemical compound.^[14].

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique that is widely used in various chemical and biochemical laboratories.

The limitation of the method is that it can only determine the structure of proteins weighing around 12,000. NMR spectroscopy does not produce efficient readable spectra for larger proteins above about 13000-15000.

Therefore, advances in multidimensional spectroscopy and isotopic labeling experiments may pave the way for a better understanding of protein structure and function.

In addition, improvements in software algorithms will help smooth out the complicated process of resonance assignment and molecular structure design.

1. Wuthrich Kurt (1990). Determination of the structure of proteins in solution by NMR spectroscopy.Das Journal of Biological Chemistry, 265(36),22059-22062.
2. Fan, T.W.-M. e Lane, A. N. (2016).Applications of NMR spectroscopy in systems biochemistry. Advances in Nuclear Magnetic Resonance Spectroscopy, 92-93, 18-53.DOI:10.1016/j.pnmrs.2016.01.005
3. Uversky, VN and Dunker, AK (2012).3.9 Intrinsically disordered proteins. Integral Biophysics, 170-211.DOI:10.1016/b978-0-12-374920-8.00312-x
4. Hinds, M.G. e Norton, R.S. (1997).NMR spectroscopy of peptides and proteins. Molecular Biotechnology, 7(3), 315-331.DOI:10.1007/bf02740822
5. Wüthrich, K. (2001).Structural Biology of Nature, 8(11), 923–925.DOI:10.1038/nsb1101-923
6. Keniry, MA & Carver, JA (2002). NMR spectroscopy of large proteins.Annual reports on NMR spectroscopy, 31–69. DOI:10.1016/s0066-4103(02)48003-9.
7. Parlak Y, Güzeler N (2016). Applications of nuclear magnetic resonance spectroscopy in food.Recent research in the journal Nutrition and Food Science;4. DOI:http://dx.doi.org/10.12944/CRNFSJ.4.Special-Issue-October.22
8. https://www.sciencedirect.com/topics/earth-and-planetary-sciences/spectroscopy
9. http://tesla.ccrc.uga.edu/courses/BioNMR2014/lectures/pdfs/secuencial-asignación.pdf
10. http://hiperfísica.phy-astr.gsu.edu/hbase/Nuclear/nmrcsh.html
11. https://users.cs.duke.edu/~brd/Teaching/Bio/asmb/Papers/Intro-reviews/flemming.pdf
12. http://www.nmr2.buffalo.edu/resources/edu/matr/nmr2_2004.pdf
13. https://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopy_of_proteins.
14. https://byjus.com/chemistry/nmr-spectroscopy/#:~:text=NMR%20Spectroscopy%20Principle,stimmen%20mit%20der%20Radiofrequenz%20überein.
15. https://www2.chemistry.msu.edu/facilities/nmr/900MHz/MCSB_NMR_applications.html