Understanding dsRNA interaction of dsRBD2 of DRB4 through 1H-15N HSQC
Keywords: NMR spectroscopy, chemical shift, 1H-15N HSQC experiment, J-coupling, dsRNA
Since its discovery, nuclear magnetic resonance (NMR) has become one of the most powerful form of spectroscopy both in chemistry and structural biology and a powerful technique that can be used to investigate the structure, dynamics, and chemical kinetics of a wide range of biochemical systems. Of all these spectroscopic methods this is the only one for which a complete analysis of the entire structure is normally expected. It involves the quantum mechanical properties of the central core nucleus of the atom. These properties are as local molecular environment, and their measurement provides a map of how the atoms are linked chemically, how close they are in space. The high resolution structural details of a molecule can be provided by NMR, which is unique among all other spectroscopic techniques. Due to the complexity of this technique it can be challenging for the novice to become a practicing NMR spectroscopist. One of the more powerful attributes of NMR spectroscopy is its ability to detect molecular motion in proteins and other polymers. Other methods of detecting molecular motion, such as ﬂuorescence spectroscopy, are limited by the small number of sites that can be probed and the narrow time scale over which the motion can be characterized.
What is Spectroscopy?
Spectroscopy is the study about the investigation and measurement of spectra produced when matter interacts with or emits electromagnetic radiation. It means that when the electromagnetic waves are radiated to the sample or matter then it either absorb or reflects or transmit or it can simply pass through the matter. If the energy gap (according to quantum mechanics, energy is discrete) is equal to the incident energy of photon then it absorbs or molecules go from ground energy level to higher one. Based on the response to light whether it reflects, absorb or transmits we can figure out what type of matter it is and different types of properties of that matter.
The net (total) spin of the nucleus is equal to the sum of spins of the nucleons after pairing them up. The net spin can be zero, half-integer or an integer. Many elements in the periodic table have non-zero “nuclear spin”. These elements/isotopes are called “NMR active”. Any molecules containing such atoms (with non-zero spin) are amenable to study using NMR spectroscopy.
The spin of nucleus can be understood easily by relating it as a rotating like a tiny planet. As spin is an abstract as well as microscopic concept hence for understanding better it needs quantum mechanical concept. NMR is all about the study of nucleus that is manipulation of nuclear spins. Nucleus spin also possess angular momentum it is not because of its rotation, it’s because of its intrinsic property of spin. Bosons are the integer spin particle. Fermions are the half-integer spin particle. According to the modern physics there are three particles “laptons, quarks and force particles” by which everything in the universe is made up. Electron is also one type of lapton which have electric charge –e ( 1.6×10^-19 C) and spin -1/2. Quarks are relatively heavier particle than laptons. Mostly there are six types of quarks in nature and in between three of them are +2e/3. The other three quarks have electric charge –e/3 as shown in figure.3.1. The force particle is different from these particles. Force particle is made up of photon which is the origin of electromagnetic wave. The photon has spin=1 and have no mass and zero electric charge. When three quarks stuck together by gluon then neutron and proton forms. The neutron is composed of three quarks: two with charge –e/3 and one with charge +2e/3 as shown in fig.3.1. Hence as its name suggests it has zero charge. But it has spin -1/2 and this spin is due to the combination of quarks spins.
The proton is also made up of three quarks but it has different combination as that of neutron. It consists of two quarks having +2e/3 and the other one has charge –e/3. That is why proton has +e electric charge and having spin -1/2.
Most nuclei possess spin and every spinning nucleus have nuclear magnetic momentum (µ), characterized by nuclear spin quantum number (I). Nuclei with odd mass numbers have half-integral spin quantum numbers (I = n/2; e.g., 1H, 15N, 13C, etc.). Nuclei with an even mass number and an even atomic number have spin quantum numbers equal to zero (I = 0; e.g., 12C, 16O, 32S, etc.). Nuclei with an even mass number and an odd atomic number have integral spin quantum numbers (I = n; e.g., 2H, 14N, 10B, etc.).
In the presence of an external magnetic ﬁeld, degeneracy in the nuclear spin levels is observed, which is known as ‘Zeeman effect’. Nucleus with spin I is (2I+1)-fold degenerate. Spins with I = 0 do not display Zeeman Effect and are known as NMR inactive. Nucleus with I > 1/2 (known as ‘quadruple nuclei’) possess electric quadruple moments arising from non-spherical nuclear charge distribution and also the life time of quadruple nuclei is short that further lead to severe broadening of spectra.
Nuclei with spin 1/2 results in two energy levels in the presence of magnetic ﬁeld and they are of great importance in NMR spectroscopy. For biomolecular NMR, important nuclei with spin 1/2 are: 1H, 15N, 13C, 19F and 31P.
Materials or substances which interact with magnetic fields are called magnetic materials or magnetic substances. This magnetic interaction can be expressed in terms of magnetic moment μ. Since there is an interaction there will have energy and that energy will be decided by the orientation of the magnetic field B and magnetic moment μ.
Emag = -μ.B (1)
In the above equation the negative sign indicates that if the direction of magnetic moment and magnetic field are in antiparallel to each other then it will have high magnetic energy and if the direction of both are in parallel to each other then it will have low magnetic energy.
As we know in universe every object wants to be stable similarly in the case of magnetic energy to minimize its energy an object tries to align along its external magnetic field and that is the principle of compass needle having permanent magnetic moment μ.
What is the origin of magnetization?
Mainly there are three sources for this: First-when electric current circulates it produces magnetic field, second-the electron which possesses magnetic moment and third the nucleus which also possesses magnetic moment. The first on is easily understood because as we know that if electric current is made to flow in a loop of wire then there is generated magnetic field. But the second and third case is difficult to understand because it’s a microscopic phenomenon. Electrons and nuclei have intrinsic magnetic moment and it is not because of its rotation.
There is closely link between spin and magnetization. Spin angular momentum and magnetic moment are proportional to each other:
μ = γS (2)
For atomic nuclei, the proportionality constant γ is called the gyromagnetic ratio. Unit is rad.sec-1.T-1.
The gyromagnetic ratio may have either sign. For particles with a positive value of γ, the magnetic moment is parallel to the angular momentum. For particles with a negative value of γ, the magnetic moment is opposite in direction to the angular momentum. As shown in fig.3.1B.
Rotating object produces an angular momentum and angular momentum is a vector quantity. There is also a spin angular momentum and its direction is called spin polarization axis. In the absence of external magnetic field the spin polarization shows all possible directions. But in the presence of external magnetic field they are aligned towards the external magnetic field as shown in fig.3.1C
Also for nucleus, same case is applicable that is its magnetic moment will be either in the same direction of spin polarization (for nuclei with γ>0) or in the opposite directions to the spin polarization (for nuclei with γ<0).
Since any spining motion will have both angular momentum as well as magnetic moment. Due to in presence of angular momentum the motion will be very dynamic in nature. To understand spining motion we are taking an example of child’s spinning top as shown in fig.3.1D.
In that figure we can see that if the axis of spin is exactly in the verticle position then it has the stable position. But whenever if the axis displaces from its verical position on that instant gravitational force and its reactions of the ground, pull the top towards the ground then the direction of the rotation changes. Even acting these forces if the top is rotating fast enough the top does not fall over immediately. That is all type of motion is described in classical physics.
The frequency of precession (ω0) is proportional to the magnetic field (B0). That can be written as
ω0 = -γB0 (3)
where B0 is the magnetic field and γ is the gyromagnetic ratio and for nuclear spins, it is called the nuclear Larmor frequency. As shown in the above equation Larmor frequency is proportional to the magnetic field and gyromagnetic ratio (γ) is the proportionality constant.
The Larmor frequency can be positive or negative values. If it is positive then precession is in the clockwise and if it is negative then the precession is in anticlockwise when looking upstream with respect to the direction of the magnetic field. For example few nuclei like 13C and 15N have positive and negative value of gyromagnetic ratio as shown in the fig.4A.
Almost all NMR spectrometer employ superconducting magnets. The superconducting coils are made up of neobium-tin alloy (for high strengths). These wires are wound miles long with uniform diameter throughout. The magnet is rested on vibration free legs to avoid vibrations from the ground to be transferred to the magnet. The higher the field strength, higher is the sensitivity of NMR (sensitivity varies as B03/2). The magnetic field is not constant and slowly drifts with time. Typically drift rates is 8-10 Hz per hours.
Its basically two types:-
- Room temperature (RT) probe, the RF coil and other parts are at room temperature.
- Cryogenic probes, the RF coil and other parts are cryo-cooled to 15-25K. Reduces thermal noise leading to increase S/N (signal to noise ratio).
The probe is that part of the NMR spectrometer system, which is used to transmit RF signals to the sample and receive the emitted RF signals from the sample. The RF coils are constructed in such a manner that they wrap around the sample. Usually there will be two layers of the coil. The inner coil is used for 1H and the outer coil for other nuclei. In many of the probes, the coils can be tuned to multiple nuclei. The probe has temperature probes to moniter the temperature around the sample.
Tuning the RF coil to the right frequency- RF coils used in NMR spectrometer need to be tuned for the specific sample being studied. The RF coil has a bandwidth or specific range of frequency at which it resonates. When we place a sample in an RF coil, the conductivity and dielectric constant of the sample affect the resonance frequency. If this frequency is different from resonance frequency of the nucleus that we are studying, the coil will efficiently set up the B1 field nor efficiently detect the signal from the sample. We will be rotating the magnetization by an angle less than 90o when think we are rotating by 90o. This will produce less transverse magnetization and less signal. Furthermore, because the coil will not be effectively detecting the signal, signal to noise ratio will be poor during NMR experiments.
Magnetic field is not uniform that is why molecules in different position will experience different field stength as shown in the figure. After that when the magnetic field is uniform then molecules in different positions will experiences same field strength as shown in the fig.5.5A.
The process of ‘shimming’ involves adjusting the current in shim coils till a homogenous B0 is obtained across the sample. A magnetic field gradient varies with position in the NMR tube as shown in the fig.5.5B.
The external magnetic field always drifts with time (e.g. 5Hz/hr). There can also be change in the magnetic field due to reasons like involvement of the heavy metal object in the vicinity. The field-frequency lock system is used to lock the external magnetic field at a given value during the course of the experiment. This is done by using a lock coil attached to the probe which will moniter the deuterium resonance and adjust the current in the coil to keep the deuterium resonance shifts. The deutrium resonance is observed as a dispersive signal.
If the lines moves in the left direction then current is added in the lock coil to bring the pic back to resonance. If the line moves right direction then current is reduced in the lock coil to bring the pic back to resonance as shown in the given figure 5.6A.
ADC is analog to digital converter, a device which digitizes an analog. The dynamic range of ADC is measured in bits. Modern day spectrometer have 16 bits ADCs.
In a molecule, there are several atoms, each having their own nucleus, each nucleus has a different electron cloud associated with it, these electron clouds circulate and induces opposite magnetic field to the external magnetic field (Lenz’s law) as shown in figure 5.8A.
The effective field experienced by the field as shown in the figure is
Where Bloc is the local magnetic field,
σ is the constant,
B0 is the applied magnetic field,
Bind is the induced magnetic field.
In any molecules, a given nucleus (or an atom) is surrounded by a cloud of electrons with specific density. These electrons (moving charges) exert a magnetic field at the nucleus which is often opposite in direction to the main magnetic field. Thus the effective field felt by the nucleus is reduced from B0 by a factor which depends on the density of the electrons around the nucleus.
The precessional frequency (ω = γB0) is not same for all nuclei in the molecule and depends on the electron density surrounding it (i.e. the chemical environment of the nucleus). As shown in the figure 5.8B. Chemical shift value is based on a reference. It does not change with field strength. (e.g. its value at 300 MHz will be same as at 700 MHz).
J - Coupling
When peaks of NMR spectrum are closely examined, it reveals fine splits in the peaks. The peak appears as multiplet of peaks as shown in fig.5.9A. This phenomenon arises due to “J-coupling” or “spin-spin interaction” is between nuclei that are separated by one, two or three covalent bonds. J-coupling (also known as scalar coupling) is mediated by the covalent bonds separating the two nuclei. Hence, it is also called “through-bond” interaction. Like chemical shift, J-coupling strength does not change with change in magnetic field strength (i.e. its value at 300MHz will be same as at 600MHz). Scalar couplings can be observed for up to 3 to 4 bonds, and conventionally represented by, nJAB.
To improve the sensitivity of NMR signal for nuclei of low-abundant and low gyromagnetic ratio the INEPT experiment was developed. There are so many nuclei which have very poor inherent sensitivity as a result of low natural abundance or their low gyromagnetic ratio. Among these nuclei 15N and 13C are noted, now extensively used for the simplification of proton spectra of biologically important macromolecules. As noise increases as the square root of the frequency, sensitivity is roughly proportional to γ5/2. As shown in the equation (5) below.
signal/noise ∝ |γ|5/2(B0)3/2 (5)
The net effect is the non-selective polarization transfer from protons to X nuclei with the appropriate 1H-X coupling that is polarization transfer that involves J-coupling. It’s the building block of several experiments such as HSQC. The basic transferring diagram is shown below in figure 8. Size of the observable signal depends on the size of the magnetization. A higher γ nucleus has a larger equilibrium magnetization. INEPT enhances sensitivity by transferring equilibrium magnetization from higher γ nucleus to lower γ one (e.g. 1H to 15N or 13C). Now from the above equation (5), residue specific chemical shift perturbation was calculated and a histogram was plotted (fig.10.1B)
1H-15N HSQC Experiment
The HSQC experiment is widely used for recording one-bond correlation spectra between 1H and 15N, with proton being the observed nucleus. The vast majority of such spectra are recorded on molecules of biological interest (peptides, proteins and nucleic acids) which can quite easily be enriched in 15N. Using 1H-15N HSQC experiment, bulk magnetization is developed on 1H, then transferred from 1H to 15N nucleus via 1J-coupling, chemical shift of nitrogen is allowed to evolve and the magnetization is transferred back to hydrogen for detection.
Now the HSQC pulse sequences are shown in figure 9A. The sequence works by first transferring magnetization from the I(1H) spin to the S(15N) spin using the same methods as in INEPT and because the magnetization associated with the insensitive spins is transferred back to the attached proton by the same mechanism that is why it is also referred to as reverse-INEPT transfer. The S spin magnetization then evolves for t1, during this time it acquires a frequency label according to the offset of S. Finally this magnetization is transferred back to I, where it is observed. The resulting spectrum thus has peaks centered at the offset of the S spin in the ω1 dimension, and at the offset of the I spin in the ω2 dimension.
Interaction of dsRBD2 of DRB4 1H-15N HSQC
Double stranded RNA binding proteins (dsRBPs) are very crucial in RNA interference. They are involved in precursor RNA recognition and formation of 20-27 nucleotides small RNA duplexes known as miRNA or siRNA. In plants precursor dsRNA molecules are recognized by DRBs and cleaved by DCLs (Dicer like proteins) into mature mi- and si-RNA. In Arabidopsis plant, anti-viral defense is mediated by DRB4:DCL4 complex. DRB4 recognizes si-RNA precursors and initiates the RNAi mechanism. DRB4 has also two RNA binding domains (DRB4D1 and DRB4D2) and one C terminal domain. In present report 13bp RNA was titrated against DRB4D2 in increasing molar ratios in order to map the RNA binding surface. 1H-15N HSQC spectrum was used to track the perturbation in interacting residues.
Residue specific chemical shift perturbation was calculated and a histogram was plotted (fig.4.1). Throughout the histogram at least three regions were found where chemical shift perturbations are very high and those residues are K84, A111, K133, K134 and E137. Chemical shift perturbations are mapped on the surface of DRB4D2 which indicates the RNA binding patch on the protein (fig.4.).
Interaction of dsRBD2 of DRB4 1H-15N HSQC
Cross peak of 1H and 15N of different amino acids were assigned from the HSQC profile of different dsRNA concentration data which helped to calculate chemical shift value for each titration. Chemical shift of each titration was saved in a list file. Final chemical shift perturbation was analyzed from sets of chemical shifts data and those were proteins: RNA molar ratio 0:1.5 list file as shown in figure 4.3.
The chemical shift deviation resulted from any changes in the environment either due to addition of ligand or temperature is calculated by the following equation:
Proteins are a category of macromolecules that has various features for the cellular functions. They are crucial by offering structural guide and appear as enzymes, carriers, or hormones. The constructing blocks of proteins (monomers) are amino acids. Each amino acid has a central carbon that is linked to an amino group, a carboxyl group, a hydrogen atom, and an R group or side chain. There are twenty amino acids, each of which differs in the R group. Each amino acid is linked to its neighbors via a peptide bond. A long chain of amino acids is regarded as a polypeptide.
Protein folding is comprised of four levels: primary, secondary, tertiary, and quaternary (shown in fig.10). Every protein has a unique sequence of amino acids. The amino acids sequence determines the secondary structure such as α helix and β pleated sheet which then adopt a 3D shape known as the tertiary structure. When two or more polypeptides mix to form the complete protein structure, the configuration is recognized as the quaternary shape of a protein. The form and function of the proteins are intricately linked; any alteration in structure induced by changes in temperature or pH may also lead to protein denaturation and a loss in function.
DISCUSSION AND CONCLUSION
Double strand RNA binding proteins interact with double stranded RNA with tri-partite contacts. Positively charged amino acids from α1 and α2 helices and conserved histidine from β1- β2 loop generally formed the contacts with dsRNA. In case of DRB4D2, K84 is present at α1, A111 is in β1- β2 which is adjacent to conserved His110 and K133, K134 and E137 are present in α2 forming the tri-partite contact with 13bp dsRNA. This RNA interaction study helped us to identify RNA binding surface on DRB4D2.
1. Malcolm H. Levitt, Spin Dynamics-Basic of Nuclear Magnetic Resonance, Wiley
2. James Keeler, Understanding NMR Spectroscopy, Wiley
3. Gordon S. Rule and T. Kevin Hitchens, Fundamental of Protein NMR Spectroscopy, Springer
4. Chiliveri, S.C., Aute, R., Rai, U. and Deshmukh, M.V., 2017. DRB4 dsRBD1 drives dsRNA recognition in Arabidopsis thaliana tasi/siRNA pathway. Nucleic acids research, 45(14), pp.8551-8563.
I would like to pay my sincere gratitude to the IASc-INSA-NASI for providing me with the fellowship to undertake this project. It was a wonderful learning experience.
I take immense pleasure in presenting this project which is a collective effort of several people.
I am very thankful to my supervisor Dr. Mandar V. Deshmukh for his ever-helpful and inspiring presence that always excited me with my project every day. I am really grateful to him for all his support and encouragement and the confidence that he had on me.
I am immensely grateful to Jaydeep Paul for his support, guidance and help during the entire duration of the project. He was always there with a smiling face to help me with everything. I thank him for all suggestion and solutions he had for my problems.
A special thanks to my lab members Ramdas Aute, Sneha Paturi, Upasana Rai, Warisha, Richa Garg and Sayali Khisty for making my time in the lab an enjoyable and educative one.
I would like to also thank BIT, Mesra for encouraging me to join this program to enrich myself.
I would also like to thank my parents for all the encouragement and supports.
I am indebted to my family and friends for their support and inspiration. I thank the almighty for his wonderful blessings.